Changes in resource perception throughout the foraging visit contribute to task specialization in the honeybee Apis mellifera

Changes in resource perception throughout the foraging visit contribute to task specialization in the honeybee Apis mellifera | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Changes in resource perception throughout the foraging visit contribute to task specialization in the honeybee Apis mellifera Emilia Moreno, Andrés Arenas This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2237679/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 May, 2023 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract Division of labor is central to the ecological success of social insects. Among foragers of the honeybee specialization for collecting nectar or pollen correlates with their sensitivity to sucrose. So far, differences in gustatory perception have been mostly studied in bees returning to the hive, but not during foraging. Here, we showed that the phase of the foraging visit (i.e. beginning or end) interacts with foraging specialization (i.e. predisposition to collect pollen or nectar) to modulate sucrose and pollen sensitivity in foragers. In concordance with previous studies, pollen foragers presented higher sucrose responsiveness than nectar foragers at the end of the foraging visit. On the contrary, pollen foragers were less responsive than nectar foragers at the beginning of the visit. Consistently, free-flying foragers accepted less concentrated sucrose solution during pollen gathering than immediately after entering the hive. Pollen perception also changes throughout foraging, as pollen foragers captured at the beginning of the visit learned and retained memories better when they were conditioned with pollen + sucrose as reward than when we used sucrose alone. Altogether, our results support the idea that changes in foragers' perception throughout the foraging visit contributes to task specialization. Biological sciences/Zoology Biological sciences/Zoology/Animal behaviour Biological sciences/Zoology/Animal physiology Biological sciences/Zoology/Entomology Biological sciences/Neuroscience Biological sciences/Neuroscience/Learning and memory Biological sciences/Neuroscience/Olfactory system Biological sciences/Neuroscience/Social behaviour Foraging division of labor Gustatory Sensitivity Honeybee (Apis mellifera L) Foraging bout Task specialization Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Division of labor is a key feature of social insects based on the performance of groups of specialized individuals that perform several activities simultaneously, enabling colonies to function efficiently. This phenomenon is explained by the response threshold model ( 1 ), which states that individuals differ in their sensitivity (and therefore in their responsiveness) to biologically relevant stimuli associated with specific tasks, leading to the emergence of division of labor ( 2 , 3 ) Thus, differential responsiveness, or sensitivity, to stimuli that act as a positive (e.g., food) or a negative (e.g., noxious events) reinforcement affects individual learning performances ( 4 – 6 ). Pollen (protein supply) and nectar (carbohydrates supply) are the main stimuli that motivates the foraging behavior of the honeybee Apis mellifera ( 7 ), and both act as reinforcements during the learning process ( 8 ). Even though both resources are generally available as reward in flowering plants at the same time ( 9 ), honeybee foragers specialize in collecting either pollen or nectar. Foraging specialization is linked to differences in sucrose sensitivity and it is probably the best studied case to assess variations in behavioral responsiveness in the context of division of labor ( 10 ). However, at first glance, findings on honeybee behavior are not consistent with the hypothesis stated in the response threshold model, as nectar foragers are less sensitive to sucrose (i.e. a major compound of nectar) than pollen foragers. By testing bees captured arriving at the hive entrance, it has been shown that pollen foragers are more responsive to a broad spectrum of sucrose concentrations than nectar foragers, which mainly respond to highly concentrated sucrose solutions. Such perceptual difference has been in turn interpreted as adaptive, as low sucrose sensitivity among nectar foragers may bias searching for productive sources that provide the colony with a higher energy gain ( 11 , 12 ). Nevertheless, differences in sucrose responsiveness do not fully explain why foragers that are highly sensitive to sucrose are prone to collect pollen. In some cases, sucrose sensitivity has also been shown to correlate with behavioral responses triggered by other stimuli such as odors ( 13 ), light ( 14 ) and gustatory stimuli (e.g., those available in pollen; see 15). Pollen foragers learn faster and retain memories better than nectar foragers when pollen is used as reward ( 16 ) and when odors are presented at low intensities ( 13 ). High gustatory and olfactory sensitivity might enable pollen foragers to better assess pollen, which consequently enhances learning of environmental cues and foraging efficiency ( 13 ). Thus, the hypothesis that sensitivity to sucrose accounts for sensitivity to a wider range of foraging-related stimuli could explain why pollen foragers are more sensitive to sucrose than nectar foragers ( 17 ). However, sucrose responsiveness does not always correlate with the behavioral responses (i.e., sensitivity to an electric shock in 18), indicating that sucrose perception does not account for responsiveness to stimuli in every sensory modality or behavioral context. Sucrose responsiveness has a genetic basis, and it is affected by the environmental conditions and by the internal state of the bee. Sensitivity to sucrose varies with age, caste, sex, foraging experience, feeding status ( 19 , 20 ), season ( 21 ), stress (handling), hormone levels, and exposure to pheromones ( 22 ). So far, gustatory perception in nectar and pollen foragers has been mostly studied in foragers arriving at the hive entrance, but not in bees foraging at the food source. Here, we hypothesize that foragers' sucrose perception is not the same throughout the foraging visit or at different stages of the foraging cycle, as it might be influenced by the interactions among internal and external factors such as genetic predisposition, motivational state, sugar satiety level, and perception of contextual cues from the food source and from the nest. We expect that changes in resource sensitivity throughout the foraging visit translate into changes in the acquisition and memory retention. We showed that the phase of the foraging visit (i.e. arrival or departure from a source) interacts with foraging specialization (i.e. predisposition to collect pollen or nectar) to modulate sucrose and pollen sensitivity in foragers. In concordance with previous studies, pollen foragers presented higher sucrose responsiveness (lower response thresholds) than nectar foragers at the end of the visit. On the contrary, and consistently with the response threshold model, we observed that pollen foragers were less responsive than nectar foragers when they arrived at the food source. Furthermore, context drastically influenced sucrose mediated response as we showed that free-flying bees were less likely to accept sucrose solution when they were collecting pollen in a feeder than when they returned (loaded with pollen) to the hive. Pollen perception also changes throughout the foraging visit. Pollen foragers captured when they arrived at the source learned and retained memories better when we used pollen + sucrose as reward during a conditioning than when we used sucrose alone. Such a difference was not detected among departing bees, indicating that pollen contribution as a reward is more important at the beginning than at the end of the foraging visit. Altogether, our results support the idea that resource perception changes throughout the foraging visit contributes to foraging specialization. Results Changes in sucrose responsiveness of foragers are influenced by foraging specialization and by the phase of the foraging visit. To evaluate sucrose responsiveness, we captured foragers arriving or departing from an artificial feeder with either sucrose solution or crushed bee-collected multifloral pollen (Fig. 1 A). Our analysis showed that both the forager type and the phase of the foraging visit affect sucrose responsiveness (χ²=75.89, df = 1; p < 0.001). Pollen foragers presented higher sucrose responsiveness (lower response thresholds) than nectar foragers at the end of the visit, as we detected significantly higher PER proportions throughout the entire range of evaluated sucrose concentrations (Fig. 2 ). On the contrary, and consistent with the prediction of the response threshold model, we observed that pollen foragers were less responsive than nectar foragers when they arrived at the foraging station (Fig. 2 ). Within forager types, pollen foragers were more sensitive to sucrose at the end of the visit (z ratio=-7.830, p < 0.0001), whereas nectar foragers showed higher sucrose responsiveness at the beginning (z ratio = 3.935, p < 0.0005). Changes in sucrose acceptance of free-flying pollen foragers are influenced by the phase of the foraging cycle. To estimate sucrose acceptance at different stages of the foraging cycle, we tested free-flying bees while they were gathering pollen in an artificial feeder or when they had returned to the hive (loaded with pollen after recollection; Fig. 1 B). At the pollen feeder, we observed that only a small proportion of foragers (0.14) accepted a 40% (w/w) sucrose solution, while almost all foragers evaluated immediately after entering the hive accepted it (0.98). Interestingly, at the feeder, the proportion of bees that responded to sucrose solution did not differ from the proportion of bees that responded to water (z ratio = 2.544 p = 0.534). On the contrary, the differences between sugar and water acceptance were highly significant inside the hive (z ratio = 3.751, p = 0.001). Changes in learning and memory of pollen foragers are influenced by the stage of the foraging visit and by using pollen as reinforcement. Given that a higher sensitivity to a stimulus that acts as a reward translates into an improvement in learning and memory retention (4–6; 18), we evaluated pollen perception by studying how bees learn and retain memories established during a conditioning with pollen as a co-reinforcement of sucrose ( 16 ). To test how pollen perception changes as a function of the phase of the foraging visit, we olfactory conditioned the PER of pollen foragers arriving at or departing from a pollen feeder using pollen + sucrose (double reinforcement) or sucrose alone as rewards (Fig. 1 C). Our analysis showed that the PER proportion was influenced by both the moment of the foraging visit and the reinforcement type (χ²=17.4799, df = 2; p = 0.00016; Fig. 4 ). For the extinction trials, our analysis detected a triple interaction between the stage of the foraging visit, the reinforcement type, and the trial (χ²=7.9720, df = 2; p = 0.018). Using the double reinforcement (pollen paired procedure or PPP), we observed that pollen foragers acquired and retained olfactory memories equally well when they arrived and when they departed from the source (adqui: z ratio = 0.145, p = 1; exti: z ratio=-0.733, p = 0.977). However, when the odor was paired with sucrose but uncoupled with the pollen presentation (pollen unpaired procedure or PUP), we observed that both acquisition and extinction performance was lower when they arrived than when they departed (adqui: z ratio=-4.212, p = 0.004; exti: z ratio=-5.002, p = < 0.001), indicating that pollen contribution as a reward is more important when bees arrive at the source than when they depart from it. Moreover, memory retention of bees captured upon arrival under the control procedures (sucrose + cellulose or CPP) improved in comparison with the unpaired conditioning (z ratio=-3.350, p = 0.0105), but it was not high enough to reach the PER proportions obtained with pollen in the PPP conditioning (z ratio=-3.123, p = 0.0221). Our results showed that cellulose worsens the performance of pollen foragers that departed from the feeder. Discussion Previous studies demonstrate the relationships between responsiveness of worker honeybees to sucrose and their foraging behavior ( 11 , 12 ). Here, we show that the timing of the foraging visit is important to the division of labor in the honeybee, as the perception of sucrose that affects foraging specialization increases or decreases throughout the foraging visit according to the bees´ foraging predisposition. Similarly, we found that sucrose acceptance varied greatly according to the phase of the foraging cycle, highlighting the idea that reward perception is not fixed but rapidly modulated by the context. Moreover, the evidence indicates that the sucrose and pollen sensitivity of pollen foragers varies in opposite directions throughout the foraging visit. The response threshold model has been extremely influential to explain division of labor and its relation to colony organization. Although specialization in nectar or pollen foraging remains as the best-studied case of how variations in behavioral responsiveness can result in task specialization, the predictions of the model have also been tested in other social insects ( 23 ). In colonies of bumblebees ( Bombus terrestris ), sucrose responsiveness tested in nectar foragers (capture during collection) was similar to pollen foragers, but higher than in bees that collected both resources, suggesting that foragers that are specialized in collecting nectar are more sensitive to sucrose than those with a more diversified collecting behavior ( 24 ). Differences in sensitivity to stimuli other than taste are also important for the division of labor. For example, differences in sensitivity to floral odors have been found between pollen and nectar foragers, with the former group being better at detecting and learning odors presented at low concentrations than nectar foragers (e.g. odors emitted by pollen; see 13). The hygienic behavior of the honeybees is also critical to understand the sensitivity to odors in honeybees. Workers have the ability to detect, uncover, and eliminate infected or parasite broods. In colonies considered “hygienic” such responses take less time than in “non-hygienic” colonies ( 25 , 26 ). Because workers from hygienic colonies exhibit higher detection and responsiveness to abnormal brood odors, we may assume that sensitive workers on non-selected colonies are those who will respond first to the infected or parasitized preimagos, which agrees with the prediction of the response threshold model. Studies in defensive behavior also show that when the hive entrance is disturbed, older honeybees are more likely to exhibit defensive behaviors in contrast to younger workers ( 27 ), a response that correlates with the increased olfactory sensitivity to the alarm pheromone in older bees ( 28 ). Yet, it has been observed that guards have lower learning performances during aversive conditionings in comparison with foragers when exposed to electric shocks ( 18 ), not fitting the prediction of the model. Therefore, given that multiple evidence both supports and contradicts that specialized individuals are the most sensitive to the task-related stimuli, more experiments on their individuals assessed in their worksites are needed to better understand how the interaction between learning, responsiveness to reinforcement, and social organization drives division of labor. In honeybees, the majority of investigations established differences in the sucrose responsiveness between nectar and pollen foragers that returned to the nest after collecting, but only a few studies analyzed the responses of foragers that departed from the hive, presumably to initiate a foraging trip ( 11 , 12 ). Using exiting foragers derived from artificially selected high- and low-pollen-hoarding strains, Page et al., (1998) found that bees from the high-pollen-hoarding strain (estimated to be composed of 40% pollen and 60% non-pollen foragers) presented higher responsiveness than bees from the low-pollen-strain (96% were non-pollen foragers). Although this experiment did not directly evaluate pollen and nectar foragers, it allows us to compare responsiveness of foragers incoming and exiting the hive. Page and coworkers showed that sucrose responsiveness of incoming "nectar foragers” decreased about 25% compared to exiting “nectar foragers”, but that incoming pollen foragers increased by 47% compared to exiting pollen foragers. This evidence is consistent with our results and supports the idea that there is an interplay between the stage of the foraging cycle and foraging predisposition, as the responses of the two groups varied in opposite ways. In another study, Pankiw et al. (2001) tested the sucrose responsiveness in foragers leaving the hive searching for a pollen source and returning to the hive loaded with pollen. They found that pollen collection did not affect the PER; however, the responsiveness of foragers that arrived at the hive was lower than those foragers exiting the nest. While we would expect that incoming foragers show a similar internal state than foragers arriving at a food source, several factors, including the social context, interactions and/or features of the food source may be critical in adjusting/modulating the bees’ sensitivity. At the food source, perception of innate cues, such as pollen odors, but also learned cues (e.g., odors, colors or shapes) might set the bees into a "foraging mode or state" that does not persist once the individual reaches the nest. Considering that in our experiment bees had previous experiences with the sources (i.e. feeders were available in previous days) and given that memories can alter gustatory perception ( 29 ), we believe that evoking memories established with pollen or sugar rewards are essential to contextualize the bees' behaviors. While the presentation of learned odors associated with nectar sensitizes bees by increasing the probability to extend the proboscis ( 30 ), stimuli learned with pollen could act the other way around, inhibiting or decreasing the probability of response. This idea is supported by the fact that learned odors at pollen sources bias e.g., the orientation of foragers, but do not induce the extension of the proboscis during an olfactory conditioning ( 31 ). Further experiments comparing the changes in the gustatory responsiveness of single foragers through different stages of the foraging cycle (including arriving and departing from the food source, during recollection, and incoming and exiting the hive) will allow us to better understand reward perception during foraging. We could also consider that when we capture and move bees in the laboratory, we interfere with the mode/state of the bees, triggering a set of other behaviors, such as begging or offering liquid food for refueling or exchanging information ( 32 ). In addition, we cannot rule out possible changes in perception associated with the stress of being immobilized in the restraints. Interestingly, during the acceptance trials with free flying pollen foragers (exp. 2; Fig. 3 ), sucrose acceptance was extremely low (a response proportion of 0.18) compared to those elicited for similar concentrated solutions in restrained bees (0.66). These differences reflect that the experimental conditions used to estimate bee’s gustatory sensitivity matter and could lead us to erroneous conclusions if we do not take these conditions into account. Such difference is consistent with a stress-reduced assessment and with the idea that the cues that surround the sources impact in resource perception. In this context, we know that pollen volatiles, such as those available in exp. 2, can be very functional in contextualizing the foraging site, as these odors are used by bees as indicators of source productivity ( 33 ). There are fundamental differences in how bees collect and transport nectar and pollen. Foraging trips involve visiting hundreds of flowers to collect a few microliters of nectar or a few micrograms of pollen before returning to the hive to unload, and then depart again for another foraging trip ( 7 ). Once the nectar is ingested, it is stored in a portion of their digestive tract (= the crop), where it is transported to the hive. On the contrary, pollen is agglutinated during recollection and transported in specialized structures on the hind legs, the corbiculae ( 34 ). In addition, pollen foragers use nectar to aggregate pollen grains into their corbiculae. Inside the hive, foragers consume honey as fuel before exiting the nest, so technically they do not need to ingest food during foraging activity. However, bees can use stored nectar from the crop to provide an immediate energy supply if they face high energy demands ( 35 ). Because unlike other insect groups, honeybees cannot use protein constituents such as proline to fuel their flight muscles ( 36 ), pollen foraging activity might be highly restricted to the initial amount of nectar that they can store in their crop, which may limit the duration of the visit and the amount of pollen that foragers can collect. Hence, it is likely that pollen and nectar foragers greatly differ in their sugar satiety level along the foraging cycle. While both groups might have similar satiety levels upon arrival at the food sources, pollen collection may generate substantial higher sugar demand. Consistent with the fact that sucrose responsiveness varies due to the nutritional status of foragers it has been observed that both groups of foragers significantly reduce their sensitivity to sucrose after feeding ( 11 , 12 ). Hence, the differences we detected between arriving and departing nectar and pollen foragers could be explained, at least in part, on the basis of changes in the satiation levels. Furthermore, crop filling affected response thresholds directly and independently of sucrose concentration, and its effect is instantaneous ( 12 ). However, pollen foragers and non-pollen foragers still differed in their responses to sucrose after feeding, suggesting that foraging predisposition plays an important role in differential responses ( 11 ). Furthermore, differences in satiety level between pollen and nectar foragers could be accentuated due to the stress we generated during the testing procedures ( 22 ), with nectar foragers being less affected than pollen foragers due to their higher supply of nectar in the crop. In a recent experiment, in which gene expression of octopamine receptors was obtained from foragers captured immediately after landing at feeders, showed that there was an overall higher expression for receptor gene AmoctαR1 in the brain of pollen than nectar foragers ( 37 ). Interestingly, pollen and nectar foragers captured at the end of their foraging bout did not show differences in AmoctαR1 expression, but for Amtyr1 in the suboesophageal ganglia. A qualitative comparison of the expression levels of AmoctαR1 gene suggests that its expression is high in the brain of pollen foragers at the beginning of the foraging trip but down regulated as the foragers become satiated and ready to abandon the feeder. Once inside the hive, this receptor might be upregulated again, driving the bees to resume pollen foraging. Hence, we speculate that changes in octopamine receptor expression relate to different phases of the pollen foraging bout ( 37 ). Although most studies focused on sucrose responsiveness, a few experiments have also measured gustatory sensitivity to pollen ( 11 , 15 ). Page and coworkers (1998) observed that pollen foragers were more likely to show the PER than nectar foragers when they were stimulated using pollen loads. Interestingly, they noticed that such differences in pollen perception became non-significant after feeding the bees with sucrose, suggesting that differences in bees' responses were triggered by sugars of pollen. Using the procedure of co-reinforcement of sucrose and pollen, we showed that foragers returning to the hive loaded with pollen learned and retained olfactory memories better than foragers captured while collecting nectar from an artificial feeder ( 16 ). Our results show that upon arrival at the food source, pollen foragers improved their performance if the conditioning included the paired presentation of pollen and sucrose as reward. We did not find differences in learning performance with pollen when bees departed from the pollen source. Nevertheless, pollen seems to be perceived as a negative (aversive), rather than appetitive, reinforcement. This suggests that the contribution of pollen as a reward is relevant during foraging but not when bees prepare to leave from the foraging site. It is interesting to note that cellulose presentation also improved the performance of bees arriving at the source (evidenced mainly during memory retention), suggesting that a tactile stimulation with inert particles similar in size to pollen grains is also perceived as an appetitive stimulus. In contrast to previous findings that showed a positive correlation between sucrose and pollen sensitivity ( 11 , 15 ), our results indicate that pollen foragers that arrived at the sources are very sensitive to pollen but no to sucrose. Even when bees can extend their proboscis by pollen stimulation ( 11 , 15 , 38 ), PER may not be the most appropriate paradigm for assessing responses related to pollen foraging, as this is a response closely associated with ingestion of liquid food. Therefore, we do not rule out the possibility that there are other procedures by which we can further explore pollen sensitivity in honeybees. Methods Study site. We carried out experiments 1 and 3 during the summer seasons of 2020–2021 and experiment 2 during the summer season of 2019 in the Experimental Field of the Faculty of Exact and Natural Sciences of the University of Buenos Aires (UBA), Argentina. We performed all experiments according to the animal care guidelines of the National Institute of Health (1985) and the current laws of Argentina. Experimental bees. We tested European honeybees A. mellifera ligustica. Foragers were trained to visit a foraging station located 50m away from the apiary. We trained the bees using a 10% sucrose solution and crushed bee-collected multifloral pollen, which were offered in separate (20cm apart) ad libitum feeders located on a wooden platform (30cm x 40cm). Experiment 1. Testing sucrose perception. In this experiment, we assessed sucrose responsiveness of pollen and nectar foragers at different phases of their foraging visit. Sucrose sensitivity can be evaluated by means of the proboscis extension reflex (PER), an innate response triggered after touching the bee's antennae with a sufficiently concentrated sucrose solution ( 39 ). We stimulated the antennae of restrained bees with a series of sucrose-water solutions of increasing concentrations (0.1, 0.3, 1, 3, 10, 30 and 50%) to determine which solution elicited the extension of the proboscis. We captured bees when they arrived (once they landed on the feeder’s surface, before resource recollection) and when they departed (once they cleaned their antennae, after resource recollection). In that way, we obtained 4 different groups of foragers: i) pollen-arrival, ii) pollen-departure, iii) nectar-arrival, and iv) nectar-departure. In the laboratory, we chilled bees in the freezer until they remained immobile and carefully restrained them in harnesses that only allowed their antennae and mouthparts to move freely ( 40 , 41 ). We offered bees water with a toothpick until satiation before we placed them into the incubator (30°C, 60% RH, and darkness) for 30 to 45 minutes. We did not feed bees during trials to not modify their motivation to respond. Bees were tested in sequential order, starting from de lowest to the highest concentration of sucrose solution with an inter-trial interval of 2 minutes (Fig. 1 a). We provide bees with water between sucrose trials to prevent responses to sucrose solution caused by thirst ( 11 ). Experiment 2. Testing sucrose acceptance. We studied the extent to which manipulation and context (i.e. foraging or social context) affect foragers' acceptance of sucrose (including the ingestion of a small sample of sucrose solution). Here, we tested bees' sucrose perception in situ in individuals that were collecting pollen from a feeder and in bees entering the hive loaded with pollen corbicules after recollection. For the first situation, we trained a group of bees to collect pure crushed multifloral bee-collected pollen from an artificial ad libitum feeder and marked them with acrylic paint of different colors to identify them during successive foraging visits. We touched the antennae of individual bees with a long stick (15 cm) embedded in sucrose sn. 40% or water shortly after the bee landed and started manipulating pollen. If bees extended their proboscis, we allowed them to ingest a drop of the solution (ca. 7 µl). The trials lasted 45 min, during which each bee made 3 to 5 trips. To measure acceptance inside the hive, we trained bees obtained from an observation hive to collect pollen from an artificial ad libitum feeder containing crushed bee-collected multifloral pollen. As before, we color-marked bees at the feeder to identify them at the foraging station and inside the hive. We measured sucrose and water acceptance immediately after focal bees enter the hive (before they unloaded their corbiculae in the hive cells; Fig. 1 b). We removed the sides of the hive, which allowed us to access focal bees easily. Sucrose solution or water was randomly assigned, and each bee was presented with the same solution throughout all visits. When possible, we tested acceptance once per visit throughout all foraging visits or hive stays. Experiment 3. Testing learning and memory using pollen as co-reinforcement. We studied the differences in the acquisition and retention of olfactory memories during associative learning at the beginning and at the end of the foraging visit. Like nectar, bees also extend their proboscis when stimulated with pollen, however; this response is not stable, and bees often stop responding to pollen after a few events. Hence, we developed an alternative procedure in which we offer a simultaneous double reinforcement presenting sugar on the antennae and pollen on the tarsi of the first pair of legs. This procedure allows us to obtain stable PER responses throughout the successive training events. We olfactory conditioned bees by the presentation of the floral odor linalool (0.1 M, Sigma- Aldrich) as conditioned stimuli to both antennae complemented with sucrose-water solution (15%) as reward in the left antenna, and hand-collected kiwi pollen as reinforcement to the left first tarsi. Memories formed during 4 acquisition trials were then evaluated along 4 extinction trials that consisted in the presentation of the odor alone. We delivered the odor paired with sucrose + pollen (pollen paired procedure, PPP). To control possible sensitization produced by the effect of pollen stimulation on bees, we presented the odor paired to sucrose but not to pollen (pollen unpaired procedure, PUP). To control for mechanical stimulation, we presented the odor paired with sucrose + cellulose (cellulose paired procedure, CPP; Fig. 1 c). Despite being an inert compound, cellulose particles (which are similar in size to pollen grains) might provide a tactile stimulation that reinforces learning and memory of bees approaching the pollen source. Finally, bees that showed a spontaneous response (i.e. extending the proboscis in response to the first odor presentation) were excluded, as we cannot determine whether this is an innate response or if it indicates a prior (uncontrolled) odor- reward association. Odor delivery . To present the CS, we used an olfactometer that sent a continuous clean air flow (50ml s-1) to the head and delivered the odor through a secondary air stream (6.25ml s-1) which was injected into the main airflow through a system of valves controlled by computer. A piece of filter paper (30x3mm) was impregnated with an aliquot of the odor (4µL) and placed inside a syringe connected to the secondary air stream. Each trial lasted 55s. The valve was programmed so that it released clean air during the first 20s, followed by the odor (6s), and a final exposure to clean air for the last 29s. The last 3s of the odor presentation overlapped with the sucrose + pollen (or cellulose) presentation in the paired procedures. During the unpaired procedures, we presented pollen or cellulose 5s after the odor + sucrose presentation. We measured the PER during the first 3s of the odor presentation. Statistical analysis. For experiments 1 and 3 we used multiplicative generalized linear mixed models (GLMMs) assuming a Bernoulli distribution. When the PER occurred, we assigned values of 1, and when it did not, we assigned values of 0. In experiment 1, we analyzed the proportion of PER of restrained foragers considering forager type (a two-level factor corresponding to pollen and nectar foragers), foraging stage (a two-level factor corresponding to beginning and end), and sucrose concentration (continuous variable) as fixed effects. Individual bees and experimental days were considered as random effects. For experiment 2, we analyzed the PER proportion in response to sucrose and to water (a two-level factor) when they arrived at the feeder and when they entered to the hive (a two-level factor) by means of a binomial multiplicative generalized linear mixed model, using the “glmmTMB'' function of the ‘glmmTMB’ package (Bates et al., 2015). Experimental days were considered as random effects. In experiment 3, we analyzed the proportion of PER of restrained pollen foragers during acquisition and extinction trials considering foraging stage (a two-level factor corresponding to beginning and end), treatment (a three-level factor corresponding to the reinforcement: paired pollen, unpaired pollen and cellulose), and trial (continuous variable) as fixed effects. Each bee was considered as random effects. We used the “glmer” function of the ‘lme4’ package ( 42 , 43 ). For all the experiments, we conducted post hoc contrasts on models to assess effects and significance between fixed factors using the “emmeans'' function of the ‘emmeans’ package version 1.4 ( 44 ) with a significance level of 0.05. Declarations Acknowledgements We thank M.J. Corriale for her help with statistical analyses and W.M. Farina for the fruitful comments at the early stage of this paper. We also thank Wagner Chaves for their comments and suggestions. Funding This study was partly supported by grants from Agencia Nacional de Promoción Cientıfica y Tecnológica (PICT_2017-2688) to A. Arenas and University of Buenos Aires (UBA-20020170100078BA). Data availability statement All data generated or analysed during this study are included in this published article [and its supplementary information files]. References Beshers, S. N. and Fewell, J. H. 2001. Models of division of labor in social insects. Annual review of entomology , 46, 413. Robinson, G. E. 1992. Regulation of division of labor in insect societies. Annual Review of Entomology . 37, 637-665. doi:10.1146/annurev.en.37.010192.003225. Bonabeau E, Theraulaz G. and Deneubourg JL. 1996. Quantitative study of the fixed threshold model for the regulation of division of labour in insect societies. Proceeding of the Royal Society of London B 263: 1565–1569. Scheiner, R., Page, R. E. and Erber, J. 2001a. 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Differences in olfactory sensitivity and odor detection correlate with foraging task specialization in honeybees Apis mellifera . Journal of Insect Physiology , 141, 104416. Erber, J., Hoorman, J. and Scheiner, R .2006. Phototactic behaviour correlates with gustatory responsiveness in honey bees ( Apis mellifera L.). Behavioral Brain Research 174: 174–180. Scheiner, R., Page, R. E. and Erber, J. 2004. Sucrose responsiveness and behavioral plasticity in honey bees (Apis mellifera). Apidologie 35, 133-142. doi:10.1051/apido:2004001 Nery, D., Moreno, E. and Arenas, A. 2020. Pollen reinforces learning in honey bee pollen foragers but not in nectar foragers. Journal of Experimental Biology. 223, 230–250. Humphries, M.A., Fondrk, M.K. and Page, R.E. 2005. Locomotion and the pollen hoarding behavioral syndrome of the honeybee ( Apis mellifera L.). Journal of Comparative Physiology A , 191, 669–674. Roussel, E., Carcaud, J., Sandoz, J. C. and Giurfa, M. 2009. Reappraising social insect behavior through aversive responsiveness and learning. PLoS One , 4(1), e4197. Pankiw T, Page RE. 1999. The effects of genotype, age, and caste on response thresholds to sucrose and foraging behavior of honey bees. Journal of Comparative Physiology A, 185, 207–213. Mengoni Goñalons, C., Guiraud, M., de Brito Sanchez, M. G., and Farina, W. M. 2016. Insulin effects on honeybee appetitive behaviour. Journal of Experimental Biology , 219(19), 3003-3008. Scheiner R, Barnert M, Erber J. 2003. Variation in water and sucrose responsiveness during the foraging season affects proboscis extension learning in honey bees. Apidologie 34:67–72 Pankiw, T., Page, R.E. 2003. Effect of pheromones, hormones, and handling on sucrose response thresholds of honey bees ( Apis mellifera L.). Journal of Comparative Physiology A, 189, 675–684. https://doi.org/10.1007/s00359-003-0442-y Perez, M., Rolland, U., Giurfa, M., d’Ettorre, P., 2013. Sucrose responsiveness, learning success, and task specialization in ants. Learning and Memory. 20, 417–420. https://doi.org/10.1101/lm.031427.113. Smith, K.E., 2016. Causes and consequences of variation in learning performance in the bumblebee ( Bombus terrestris ). University of London, Royal Holloway. PhD thesis. Gramacho, K. P., and Spivak, M. 2003. Differences in olfactory sensitivity and behavioral responses among honey bees bred for hygienic behavior. Behavioral Ecology and Sociobiology , 54 (5), 472-479. DOI: 10.1007/s00265-003-0643-y Spivak, M., Masterman, R., Ross, R., Mesce, K.A. 2003. Hygienic behavior in the honey bee ( Apis mellifera L.) and the modulatory role of octopamine. Journal of Neurobiology. 55 (3), 341–354. https://doi.org/10.1002/neu.10219 . Breed, M. D., Robinson, G. E., Page, R. E. 1991 .Division of labor during honey bee colony defense. Behavioral Ecology and Sociobiology. 27:395-401 Robinson, G. E. 1987. Modulation of alarm pheromone perception in the honey bee: evidence for division of labor based on hormonally regulated response thresholds. Journal of Comparative Physiology 1. 60:61 3-19 Ramírez, G. P., Martínez, A. S., Fernández, V. M., Corti Bielsa, G., and Farina, W. M. 2010. The influence of gustatory and olfactory experiences on responsiveness to reward in the honeybee. PloS one , 5 (10), e13498. Hammer, M., and Menzel, R. 1995. Learning and memory in the honeybee. Journal of Neuroscience , 15 (3), 1617-1630. Arenas, A. and Farina, W.M. 2012. Learned olfactory cues affect pollen-foraging preferences in honeybees . Apis mellifera . Animal Behaviour . 83, 1023–1033. https://doi.org/10.1016/j.anbehav.2012.01.026. De Marco, R. J., and Farina, W. M. 2003. Trophallaxis in forager honeybees (Apis mellifera): resource uncertainty enhances begging contacts? Journal of Comparative Physiology A , 189(2), 125-134. Raguso, R.A. 2008. Wake Up and Smell the Roses: The Ecology and Evolution of Floral Scent. Annual Review of Ecology, Evolution, and Systematics, 39, 549-569. https://doi.org/10.1146/annurev.ecolsys.38.091206.095601 Winston, M.L. 1987. Biology of the Honey Bee. Harvard University Press. Blatt, J., and Roces, F. 2001. Haemolymph sugar levels in foraging honeybees ( Apis mellifera carnica ): dependence on metabolic rate and in vivo measurement of maximal rates of trehalose synthesis. Journal of Experimental Biology , 204(15), 2709-2716. Teulier, L., Weber, J.-M., Crevier, J., and Darveau, C.A. 2016. Proline as a fuel for insect flight: enhancing carbohydrate oxidation in hymenopterans. Proceedings of the Royal Society B: Biological Sciences , 283(1834), 20160333. Arenas, A., Lajad, R., Peng, T., Grüter, C., and Farina, W. 2021. Correlation between octopaminergic signaling and foraging task specialization in honeybees. Genes, Brain and Behavior , 20(4), e12718. Grüter, C., Arenas, A. and Farina, W. M. 2008. Does pollen function as a reward for honeybees in associative learning?. Insectes Sociaux. 55, 425–427. Frings, H. 1944. The loci of olfactory end-organs in the honey-bee, Apis mellifera Linn . Journal of Experimental Zoology , 97, 123–134. https://doi.org/10.1002/jez.1400970203 Kuwabara, M., 1957. Bildung Des Bedingten re exes Von Pavlovs Typus Bei Der Honigbiene, Apis mellifera . J. Fac. Sci. Hokkaido Univ. Zoology. 13, 458–464 Bitterman ME, Menzel R, Fietz A, Schafer S. 1983. Classical conditioning of proboscis extension in honeybees Apis mellifera ). Journal of Comparative Physiology A , 97, 107-119 Bates, D., Mä chler, M., Bolker, B. M. and Walker, S. C. 2015. Fitting linear mixed- effects models using lme4. Journal of Statistical Software. 67, 1-48. doi:10.18637/jss.v067.i01 Lenth, R. V. 2015. The case against normal plots of effects. Journal of Quality Technology . 47, 91-97. doi:10.1080/00224065.2015.11918111 Lenth, R. 2019. emmeans: Estimated marginal means, aka least-squares means. https:// cran.r project.org/ web/packages/emmeans. Additional Declarations No competing interests reported. Supplementary Files Rawdata.xlsx Cite Share Download PDF Status: Published Journal Publication published 19 May, 2023 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 07 Mar, 2023 Reviews received at journal 27 Feb, 2023 Reviewers agreed at journal 24 Feb, 2023 Reviewers invited by journal 23 Feb, 2023 Editor assigned by journal 28 Dec, 2022 Editor invited by journal 11 Nov, 2022 Submission checks completed at journal 11 Nov, 2022 First submitted to journal 04 Nov, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2237679","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":151319410,"identity":"24969a71-cce2-45d0-9f9e-537b27bdaaf3","order_by":0,"name":"Emilia Moreno","email":"","orcid":"","institution":"IFIBYNE","correspondingAuthor":false,"prefix":"","firstName":"Emilia","middleName":"","lastName":"Moreno","suffix":""},{"id":151319411,"identity":"123f45a8-9dd3-4d7e-ab18-2a82d445e51a","order_by":1,"name":"Andrés Arenas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYBADOQMg8QHMlCBSizFQC+MMkrQkbiBaC3/72YePC2rq0rfzL2Bs+FFxh8HgdvMDhp97cGuROJNubDzj2OHcnTMeMDb2nHnGYHDnmAFjzzM81hxIY5PmYTuQu+HGAfbHjG2HGQxuJBgw8BzArUP+/DP23zz/6tINbhxgbGb8B9KS/oHxDx4tBjfS2Jh525gTDM43ALU0gLTkGDDjs8XwxjNmad6+w4Y7ZzA2NvYcO8wjeedMwWEZPFrkzqcxfub5Vidvzn/4YMOPmsNyfLfbNz58g0cLAkgkNoAoHhBBlAZgDBGpbhSMglEwCkYeAACyKVhE1BVuNgAAAABJRU5ErkJggg==","orcid":"","institution":"IFIBYNE","correspondingAuthor":true,"prefix":"","firstName":"Andrés","middleName":"","lastName":"Arenas","suffix":""}],"badges":[],"createdAt":"2022-11-04 10:44:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2237679/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2237679/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-023-35163-y","type":"published","date":"2023-05-19T20:53:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":29036816,"identity":"4b3ced0d-a680-4ed7-928d-c2297031567e","added_by":"auto","created_at":"2022-11-14 16:10:17","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":54604,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic schedule of the experiments. A\u003cstrong\u003e) \u003c/strong\u003e\u003cem\u003eExperiment 1: Sucrose responsiveness.\u003c/em\u003e \u003cem\u003eAd libitum\u003c/em\u003e nectar and pollen feeders were located on a foraging station 50 m away from the apiary. We captured bees either at the beginning or at the end of the foraging event. We harnessed the bees and stimulated their antennae with a series of sucrose-water solutions of increasing concentrations (0.1, 0.3, 1, 3, 10, 30 and 50%) to determine which solution elicited the extension of the proboscis (PER). Bees were tested in sequential order, starting from de lowest to the highest concentration of sucrose-water solution. We provide bees with water between sucrose trials to prevent responses to sucrose solution caused by thirst. We waited an inter-trial interval of 2 minutes between each presentation. \u003cstrong\u003eB)\u003c/strong\u003e \u003cem\u003eExperiment 2: Sucrose acceptance\u003c/em\u003e. We trained a group of bees to collect pure crushed multifloral bee-collected pollen from an artificial \u003cem\u003ead libitum\u003c/em\u003e feeder and marked them with acrylic paint of different colors for identification during successive foraging visits. We tested bees' sucrose perception \u003cem\u003ein situ\u003c/em\u003e while they were collecting pollen from a feeder or when they entered the hive loaded with pollen corbicules after recollection and before pollen unloading. We contacted the antennae of individual bees with a long stick (15 cm) embedded in sucrose sn. 40% or water. If bees extended their proboscis, we allowed them to ingest a drop of the solution (ca. 7 µl). \u003cstrong\u003eC) \u003c/strong\u003e\u003cem\u003eExperiment 3: Olfactory conditioning with co-reinforcement\u003c/em\u003e. We captured bees either when they arrived or when they departed from the \u003cem\u003ead libitum \u003c/em\u003epollen feeder. We harnessed the bees. We olfactory conditioned bees by the presentation of the floral odor linalool (0.1 M, Sigma-Aldrich) as conditioned stimuli to both antennae, sucrose-water solution (15%) as reward to the left antenna and hand-collected kiwi pollen as co-reinforcement to the left first tarsi. Memories formed during 4 acquisition trials were then evaluated along 4 extinction trials that consisted in the presentation of the odor alone.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2237679/v1/5911c9da0971c16f073129ce.jpeg"},{"id":29037735,"identity":"a0ea39dc-9451-4a25-95aa-d7ef40678be9","added_by":"auto","created_at":"2022-11-14 16:18:17","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":55741,"visible":true,"origin":"","legend":"\u003cp\u003eProportion of PER of pollen (orange lines with circles) and nectar (blue lines with triangles) foragers in response to increasing concentrations of sucrose-water solution at the beginning (solid lines) and at the end (dotted lines) of the foraging event. Different letters indicate statistical differences (p \u0026lt; 0.05). Sample sizes are indicated in parenthesis.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2237679/v1/385e509a96a414ce38e53ea9.jpeg"},{"id":29037736,"identity":"d2016cb0-7468-4130-816a-749a7a8c8d7c","added_by":"auto","created_at":"2022-11-14 16:18:17","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34583,"visible":true,"origin":"","legend":"\u003cp\u003eProportion of PER of pollen foragers in response to sucrose (circles) or water (squares) during pollen recollection at the feeder (orange lines) or inside the hive (gray lines). Different letters indicate statistical differences ( p \u0026lt; 0.05). Sample sizes are FS=19, HS=20, FW=9, HW=8.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2237679/v1/630b3a44cd5ea05cbd81a536.jpeg"},{"id":29036820,"identity":"208c95bb-ff19-4729-bd79-2a645748f288","added_by":"auto","created_at":"2022-11-14 16:10:17","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":50944,"visible":true,"origin":"","legend":"\u003cp\u003eProportion of PER responses of pollen foragers during 5 acquisition (left) and 4 extinction (right) trials at the beginning (solid lines) and at the end (dotted lines) of the foraging trip. Pollen was used as reinforcement paired (PPP; orange line with circles) or unpaired (PUP;blue line with triangles) with sucrose-water solution. Cellulose (CPP; gray line with squares) paired with sucrose-water solution was used as control. Different letters indicate statistical differences ( p \u0026lt; 0.05). Sample sizes are indicated in parenthesis.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2237679/v1/8c1a740641d494a9f23acc28.jpeg"},{"id":44729774,"identity":"2982c723-b980-45c7-b931-5eb74aeff97c","added_by":"auto","created_at":"2023-10-16 21:20:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":490538,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2237679/v1/e64ca5fa-ebdf-47d1-ba02-174268785b9e.pdf"},{"id":29036818,"identity":"e3d58d7a-8a99-4fe2-bcbb-66703cc4edbc","added_by":"auto","created_at":"2022-11-14 16:10:17","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":75349,"visible":true,"origin":"","legend":"","description":"","filename":"Rawdata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2237679/v1/70bf94d29d3af85ad275ea33.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Changes in resource perception throughout the foraging visit contribute to task specialization in the honeybee Apis mellifera","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDivision of labor is a key feature of social insects based on the performance of groups of specialized individuals that perform several activities simultaneously, enabling colonies to function efficiently. This phenomenon is explained by the response threshold model (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), which states that individuals differ in their sensitivity (and therefore in their responsiveness) to biologically relevant stimuli associated with specific tasks, leading to the emergence of division of labor (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Thus, differential responsiveness, or sensitivity, to stimuli that act as a positive (e.g., food) or a negative (e.g., noxious events) reinforcement affects individual learning performances (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePollen (protein supply) and nectar (carbohydrates supply) are the main stimuli that motivates the foraging behavior of the honeybee \u003cem\u003eApis mellifera\u003c/em\u003e (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), and both act as reinforcements during the learning process (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Even though both resources are generally available as reward in flowering plants at the same time (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), honeybee foragers specialize in collecting either pollen or nectar. Foraging specialization is linked to differences in sucrose sensitivity and it is probably the best studied case to assess variations in behavioral responsiveness in the context of division of labor (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). However, at first glance, findings on honeybee behavior are not consistent with the hypothesis stated in the response threshold model, as nectar foragers are less sensitive to sucrose (i.e. a major compound of nectar) than pollen foragers. By testing bees captured arriving at the hive entrance, it has been shown that pollen foragers are more responsive to a broad spectrum of sucrose concentrations than nectar foragers, which mainly respond to highly concentrated sucrose solutions. Such perceptual difference has been in turn interpreted as adaptive, as low sucrose sensitivity among nectar foragers may bias searching for productive sources that provide the colony with a higher energy gain (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Nevertheless, differences in sucrose responsiveness do not fully explain why foragers that are highly sensitive to sucrose are prone to collect pollen. In some cases, sucrose sensitivity has also been shown to correlate with behavioral responses triggered by other stimuli such as odors (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), light (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) and gustatory stimuli (e.g., those available in pollen; see 15).\u003c/p\u003e \u003cp\u003ePollen foragers learn faster and retain memories better than nectar foragers when pollen is used as reward (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) and when odors are presented at low intensities (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). High gustatory and olfactory sensitivity might enable pollen foragers to better assess pollen, which consequently enhances learning of environmental cues and foraging efficiency (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Thus, the hypothesis that sensitivity to sucrose accounts for sensitivity to a wider range of foraging-related stimuli could explain why pollen foragers are more sensitive to sucrose than nectar foragers (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). However, sucrose responsiveness does not always correlate with the behavioral responses (i.e., sensitivity to an electric shock in 18), indicating that sucrose perception does not account for responsiveness to stimuli in every sensory modality or behavioral context.\u003c/p\u003e \u003cp\u003eSucrose responsiveness has a genetic basis, and it is affected by the environmental conditions and by the internal state of the bee. Sensitivity to sucrose varies with age, caste, sex, foraging experience, feeding status (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), season (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), stress (handling), hormone levels, and exposure to pheromones (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). So far, gustatory perception in nectar and pollen foragers has been mostly studied in foragers arriving at the hive entrance, but not in bees foraging at the food source. Here, we hypothesize that foragers' sucrose perception is not the same throughout the foraging visit or at different stages of the foraging cycle, as it might be influenced by the interactions among internal and external factors such as genetic predisposition, motivational state, sugar satiety level, and perception of contextual cues from the food source and from the nest. We expect that changes in resource sensitivity throughout the foraging visit translate into changes in the acquisition and memory retention.\u003c/p\u003e \u003cp\u003eWe showed that the phase of the foraging visit (i.e. arrival or departure from a source) interacts with foraging specialization (i.e. predisposition to collect pollen or nectar) to modulate sucrose and pollen sensitivity in foragers. In concordance with previous studies, pollen foragers presented higher sucrose responsiveness (lower response thresholds) than nectar foragers at the end of the visit. On the contrary, and consistently with the response threshold model, we observed that pollen foragers were less responsive than nectar foragers when they arrived at the food source. Furthermore, context drastically influenced sucrose mediated response as we showed that free-flying bees were less likely to accept sucrose solution when they were collecting pollen in a feeder than when they returned (loaded with pollen) to the hive. Pollen perception also changes throughout the foraging visit. Pollen foragers captured when they arrived at the source learned and retained memories better when we used pollen\u0026thinsp;+\u0026thinsp;sucrose as reward during a conditioning than when we used sucrose alone. Such a difference was not detected among departing bees, indicating that pollen contribution as a reward is more important at the beginning than at the end of the foraging visit. Altogether, our results support the idea that resource perception changes throughout the foraging visit contributes to foraging specialization.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eChanges in sucrose responsiveness of foragers are influenced by foraging specialization and by the phase of the foraging visit.\u003c/b\u003e To evaluate sucrose responsiveness, we captured foragers arriving or departing from an artificial feeder with either sucrose solution or crushed bee-collected multifloral pollen (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Our analysis showed that both the forager type and the phase of the foraging visit affect sucrose responsiveness (χ\u0026sup2;=75.89, df\u0026thinsp;=\u0026thinsp;1; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Pollen foragers presented higher sucrose responsiveness (lower response thresholds) than nectar foragers at the end of the visit, as we detected significantly higher PER proportions throughout the entire range of evaluated sucrose concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). On the contrary, and consistent with the prediction of the response threshold model, we observed that pollen foragers were less responsive than nectar foragers when they arrived at the foraging station (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Within forager types, pollen foragers were more sensitive to sucrose at the end of the visit (z ratio=-7.830, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), whereas nectar foragers showed higher sucrose responsiveness at the beginning (z ratio\u0026thinsp;=\u0026thinsp;3.935, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0005).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eChanges in sucrose acceptance of free-flying pollen foragers are influenced by the phase of the foraging cycle.\u003c/b\u003e To estimate sucrose acceptance at different stages of the foraging cycle, we tested free-flying bees while they were gathering pollen in an artificial feeder or when they had returned to the hive (loaded with pollen after recollection; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). At the pollen feeder, we observed that only a small proportion of foragers (0.14) accepted a 40% (w/w) sucrose solution, while almost all foragers evaluated immediately after entering the hive accepted it (0.98). Interestingly, at the feeder, the proportion of bees that responded to sucrose solution did not differ from the proportion of bees that responded to water (z ratio\u0026thinsp;=\u0026thinsp;2.544 p\u0026thinsp;=\u0026thinsp;0.534). On the contrary, the differences between sugar and water acceptance were highly significant inside the hive (z ratio\u0026thinsp;=\u0026thinsp;3.751, p\u0026thinsp;=\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003cb\u003eChanges in learning and memory of pollen foragers are influenced by the stage of the foraging visit and by using pollen as reinforcement.\u003c/b\u003e Given that a higher sensitivity to a stimulus that acts as a reward translates into an improvement in learning and memory retention (4\u0026ndash;6; 18), we evaluated pollen perception by studying how bees learn and retain memories established during a conditioning with pollen as a co-reinforcement of sucrose (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). To test how pollen perception changes as a function of the phase of the foraging visit, we olfactory conditioned the PER of pollen foragers arriving at or departing from a pollen feeder using pollen\u0026thinsp;+\u0026thinsp;sucrose (double reinforcement) or sucrose alone as rewards (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Our analysis showed that the PER proportion was influenced by both the moment of the foraging visit and the reinforcement type (χ\u0026sup2;=17.4799, df\u0026thinsp;=\u0026thinsp;2; p\u0026thinsp;=\u0026thinsp;0.00016; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). For the extinction trials, our analysis detected a triple interaction between the stage of the foraging visit, the reinforcement type, and the trial (χ\u0026sup2;=7.9720, df\u0026thinsp;=\u0026thinsp;2; p\u0026thinsp;=\u0026thinsp;0.018). Using the double reinforcement (pollen paired procedure or PPP), we observed that pollen foragers acquired and retained olfactory memories equally well when they arrived and when they departed from the source (adqui: z ratio\u0026thinsp;=\u0026thinsp;0.145, p\u0026thinsp;=\u0026thinsp;1; exti: z ratio=-0.733, p\u0026thinsp;=\u0026thinsp;0.977). However, when the odor was paired with sucrose but uncoupled with the pollen presentation (pollen unpaired procedure or PUP), we observed that both acquisition and extinction performance was lower when they arrived than when they departed (adqui: z ratio=-4.212, p\u0026thinsp;=\u0026thinsp;0.004; exti: z ratio=-5.002, p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating that pollen contribution as a reward is more important when bees arrive at the source than when they depart from it. Moreover, memory retention of bees captured upon arrival under the control procedures (sucrose\u0026thinsp;+\u0026thinsp;cellulose or CPP) improved in comparison with the unpaired conditioning (z ratio=-3.350, p\u0026thinsp;=\u0026thinsp;0.0105), but it was not high enough to reach the PER proportions obtained with pollen in the PPP conditioning (z ratio=-3.123, p\u0026thinsp;=\u0026thinsp;0.0221). Our results showed that cellulose worsens the performance of pollen foragers that departed from the feeder.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious studies demonstrate the relationships between responsiveness of worker honeybees to sucrose and their foraging behavior (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Here, we show that the timing of the foraging visit is important to the division of labor in the honeybee, as the perception of sucrose that affects foraging specialization increases or decreases throughout the foraging visit according to the bees\u0026acute; foraging predisposition. Similarly, we found that sucrose acceptance varied greatly according to the phase of the foraging cycle, highlighting the idea that reward perception is not fixed but rapidly modulated by the context. Moreover, the evidence indicates that the sucrose and pollen sensitivity of pollen foragers varies in opposite directions throughout the foraging visit.\u003c/p\u003e \u003cp\u003eThe response threshold model has been extremely influential to explain division of labor and its relation to colony organization. Although specialization in nectar or pollen foraging remains as the best-studied case of how variations in behavioral responsiveness can result in task specialization, the predictions of the model have also been tested in other social insects (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). In colonies of bumblebees (\u003cem\u003eBombus terrestris\u003c/em\u003e), sucrose responsiveness tested in nectar foragers (capture during collection) was similar to pollen foragers, but higher than in bees that collected both resources, suggesting that foragers that are specialized in collecting nectar are more sensitive to sucrose than those with a more diversified collecting behavior (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDifferences in sensitivity to stimuli other than taste are also important for the division of labor. For example, differences in sensitivity to floral odors have been found between pollen and nectar foragers, with the former group being better at detecting and learning odors presented at low concentrations than nectar foragers (e.g. odors emitted by pollen; see 13). The hygienic behavior of the honeybees is also critical to understand the sensitivity to odors in honeybees. Workers have the ability to detect, uncover, and eliminate infected or parasite broods. In colonies considered \u0026ldquo;hygienic\u0026rdquo; such responses take less time than in \u0026ldquo;non-hygienic\u0026rdquo; colonies (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Because workers from hygienic colonies exhibit higher detection and responsiveness to abnormal brood odors, we may assume that sensitive workers on non-selected colonies are those who will respond first to the infected or parasitized preimagos, which agrees with the prediction of the response threshold model. Studies in defensive behavior also show that when the hive entrance is disturbed, older honeybees are more likely to exhibit defensive behaviors in contrast to younger workers (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), a response that correlates with the increased olfactory sensitivity to the alarm pheromone in older bees (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Yet, it has been observed that guards have lower learning performances during aversive conditionings in comparison with foragers when exposed to electric shocks (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), not fitting the prediction of the model. Therefore, given that multiple evidence both supports and contradicts that specialized individuals are the most sensitive to the task-related stimuli, more experiments on their individuals assessed in their worksites are needed to better understand how the interaction between learning, responsiveness to reinforcement, and social organization drives division of labor.\u003c/p\u003e \u003cp\u003eIn honeybees, the majority of investigations established differences in the sucrose responsiveness between nectar and pollen foragers that returned to the nest after collecting, but only a few studies analyzed the responses of foragers that departed from the hive, presumably to initiate a foraging trip (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Using exiting foragers derived from artificially selected high- and low-pollen-hoarding strains, Page et al., (1998) found that bees from the high-pollen-hoarding strain (estimated to be composed of 40% pollen and 60% non-pollen foragers) presented higher responsiveness than bees from the low-pollen-strain (96% were non-pollen foragers). Although this experiment did not directly evaluate pollen and nectar foragers, it allows us to compare responsiveness of foragers incoming and exiting the hive. Page and coworkers showed that sucrose responsiveness of incoming \"nectar foragers\u0026rdquo; decreased about 25% compared to exiting \u0026ldquo;nectar foragers\u0026rdquo;, but that incoming pollen foragers increased by 47% compared to exiting pollen foragers. This evidence is consistent with our results and supports the idea that there is an interplay between the stage of the foraging cycle and foraging predisposition, as the responses of the two groups varied in opposite ways. In another study, Pankiw et al. (2001) tested the sucrose responsiveness in foragers leaving the hive searching for a pollen source and returning to the hive loaded with pollen. They found that pollen collection did not affect the PER; however, the responsiveness of foragers that arrived at the hive was lower than those foragers exiting the nest.\u003c/p\u003e \u003cp\u003eWhile we would expect that incoming foragers show a similar internal state than foragers arriving at a food source, several factors, including the social context, interactions and/or features of the food source may be critical in adjusting/modulating the bees\u0026rsquo; sensitivity. At the food source, perception of innate cues, such as pollen odors, but also learned cues (e.g., odors, colors or shapes) might set the bees into a \"foraging mode or state\" that does not persist once the individual reaches the nest. Considering that in our experiment bees had previous experiences with the sources (i.e. feeders were available in previous days) and given that memories can alter gustatory perception (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), we believe that evoking memories established with pollen or sugar rewards are essential to contextualize the bees' behaviors. While the presentation of learned odors associated with nectar sensitizes bees by increasing the probability to extend the proboscis (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), stimuli learned with pollen could act the other way around, inhibiting or decreasing the probability of response. This idea is supported by the fact that learned odors at pollen sources bias e.g., the orientation of foragers, but do not induce the extension of the proboscis during an olfactory conditioning (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Further experiments comparing the changes in the gustatory responsiveness of single foragers through different stages of the foraging cycle (including arriving and departing from the food source, during recollection, and incoming and exiting the hive) will allow us to better understand reward perception during foraging.\u003c/p\u003e \u003cp\u003eWe could also consider that when we capture and move bees in the laboratory, we interfere with the mode/state of the bees, triggering a set of other behaviors, such as begging or offering liquid food for refueling or exchanging information (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In addition, we cannot rule out possible changes in perception associated with the stress of being immobilized in the restraints. Interestingly, during the acceptance trials with free flying pollen foragers (exp. 2; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e), sucrose acceptance was extremely low (a response proportion of 0.18) compared to those elicited for similar concentrated solutions in restrained bees (0.66). These differences reflect that the experimental conditions used to estimate bee\u0026rsquo;s gustatory sensitivity matter and could lead us to erroneous conclusions if we do not take these conditions into account. Such difference is consistent with a stress-reduced assessment and with the idea that the cues that surround the sources impact in resource perception. In this context, we know that pollen volatiles, such as those available in exp. 2, can be very functional in contextualizing the foraging site, as these odors are used by bees as indicators of source productivity (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThere are fundamental differences in how bees collect and transport nectar and pollen. Foraging trips involve visiting hundreds of flowers to collect a few microliters of nectar or a few micrograms of pollen before returning to the hive to unload, and then depart again for another foraging trip (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Once the nectar is ingested, it is stored in a portion of their digestive tract (=\u0026thinsp;the crop), where it is transported to the hive. On the contrary, pollen is agglutinated during recollection and transported in specialized structures on the hind legs, the corbiculae (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). In addition, pollen foragers use nectar to aggregate pollen grains into their corbiculae. Inside the hive, foragers consume honey as fuel before exiting the nest, so technically they do not need to ingest food during foraging activity. However, bees can use stored nectar from the crop to provide an immediate energy supply if they face high energy demands (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Because unlike other insect groups, honeybees cannot use protein constituents such as proline to fuel their flight muscles (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), pollen foraging activity might be highly restricted to the initial amount of nectar that they can store in their crop, which may limit the duration of the visit and the amount of pollen that foragers can collect. Hence, it is likely that pollen and nectar foragers greatly differ in their sugar satiety level along the foraging cycle. While both groups might have similar satiety levels upon arrival at the food sources, pollen collection may generate substantial higher sugar demand. Consistent with the fact that sucrose responsiveness varies due to the nutritional status of foragers it has been observed that both groups of foragers significantly reduce their sensitivity to sucrose after feeding (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Hence, the differences we detected between arriving and departing nectar and pollen foragers could be explained, at least in part, on the basis of changes in the satiation levels. Furthermore, crop filling affected response thresholds directly and independently of sucrose concentration, and its effect is instantaneous (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). However, pollen foragers and non-pollen foragers still differed in their responses to sucrose after feeding, suggesting that foraging predisposition plays an important role in differential responses (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Furthermore, differences in satiety level between pollen and nectar foragers could be accentuated due to the stress we generated during the testing procedures (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), with nectar foragers being less affected than pollen foragers due to their higher supply of nectar in the crop.\u003c/p\u003e \u003cp\u003eIn a recent experiment, in which gene expression of octopamine receptors was obtained from foragers captured immediately after landing at feeders, showed that there was an overall higher expression for receptor gene AmoctαR1 in the brain of pollen than nectar foragers (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Interestingly, pollen and nectar foragers captured at the end of their foraging bout did not show differences in AmoctαR1 expression, but for Amtyr1 in the suboesophageal ganglia. A qualitative comparison of the expression levels of AmoctαR1 gene suggests that its expression is high in the brain of pollen foragers at the beginning of the foraging trip but down regulated as the foragers become satiated and ready to abandon the feeder. Once inside the hive, this receptor might be upregulated again, driving the bees to resume pollen foraging. Hence, we speculate that changes in octopamine receptor expression relate to different phases of the pollen foraging bout (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough most studies focused on sucrose responsiveness, a few experiments have also measured gustatory sensitivity to pollen (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Page and coworkers (1998) observed that pollen foragers were more likely to show the PER than nectar foragers when they were stimulated using pollen loads. Interestingly, they noticed that such differences in pollen perception became non-significant after feeding the bees with sucrose, suggesting that differences in bees' responses were triggered by sugars of pollen. Using the procedure of co-reinforcement of sucrose and pollen, we showed that foragers returning to the hive loaded with pollen learned and retained olfactory memories better than foragers captured while collecting nectar from an artificial feeder (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Our results show that upon arrival at the food source, pollen foragers improved their performance if the conditioning included the paired presentation of pollen and sucrose as reward. We did not find differences in learning performance with pollen when bees departed from the pollen source. Nevertheless, pollen seems to be perceived as a negative (aversive), rather than appetitive, reinforcement. This suggests that the contribution of pollen as a reward is relevant during foraging but not when bees prepare to leave from the foraging site. It is interesting to note that cellulose presentation also improved the performance of bees arriving at the source (evidenced mainly during memory retention), suggesting that a tactile stimulation with inert particles similar in size to pollen grains is also perceived as an appetitive stimulus. In contrast to previous findings that showed a positive correlation between sucrose and pollen sensitivity (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), our results indicate that pollen foragers that arrived at the sources are very sensitive to pollen but no to sucrose.\u003c/p\u003e \u003cp\u003eEven when bees can extend their proboscis by pollen stimulation (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), PER may not be the most appropriate paradigm for assessing responses related to pollen foraging, as this is a response closely associated with ingestion of liquid food. Therefore, we do not rule out the possibility that there are other procedures by which we can further explore pollen sensitivity in honeybees.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eStudy site.\u003c/b\u003e We carried out experiments 1 and 3 during the summer seasons of 2020\u0026ndash;2021 and experiment 2 during the summer season of 2019 in the Experimental Field of the Faculty of Exact and Natural Sciences of the University of Buenos Aires (UBA), Argentina. We performed all experiments according to the animal care guidelines of the National Institute of Health (1985) and the current laws of Argentina.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperimental bees.\u003c/b\u003e We tested European honeybees \u003cem\u003eA. mellifera ligustica.\u003c/em\u003e Foragers were trained to visit a foraging station located 50m away from the apiary. We trained the bees using a 10% sucrose solution and crushed bee-collected multifloral pollen, which were offered in separate (20cm apart) \u003cem\u003ead libitum\u003c/em\u003e feeders located on a wooden platform (30cm x 40cm).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperiment 1. Testing sucrose perception.\u003c/b\u003e In this experiment, we assessed sucrose responsiveness of pollen and nectar foragers at different phases of their foraging visit. Sucrose sensitivity can be evaluated by means of the proboscis extension reflex (PER), an innate response triggered after touching the bee's antennae with a sufficiently concentrated sucrose solution (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). We stimulated the antennae of restrained bees with a series of sucrose-water solutions of increasing concentrations (0.1, 0.3, 1, 3, 10, 30 and 50%) to determine which solution elicited the extension of the proboscis. We captured bees when they arrived (once they landed on the feeder\u0026rsquo;s surface, before resource recollection) and when they departed (once they cleaned their antennae, after resource recollection). In that way, we obtained 4 different groups of foragers: i) pollen-arrival, ii) pollen-departure, iii) nectar-arrival, and iv) nectar-departure. In the laboratory, we chilled bees in the freezer until they remained immobile and carefully restrained them in harnesses that only allowed their antennae and mouthparts to move freely (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). We offered bees water with a toothpick until satiation before we placed them into the incubator (30\u0026deg;C, 60% RH, and darkness) for 30 to 45 minutes. We did not feed bees during trials to not modify their motivation to respond. Bees were tested in sequential order, starting from de lowest to the highest concentration of sucrose solution with an inter-trial interval of 2 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). We provide bees with water between sucrose trials to prevent responses to sucrose solution caused by thirst (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperiment 2. Testing sucrose acceptance.\u003c/b\u003e We studied the extent to which manipulation and context (i.e. foraging or social context) affect foragers' acceptance of sucrose (including the ingestion of a small sample of sucrose solution). Here, we tested bees' sucrose perception \u003cem\u003ein situ\u003c/em\u003e in individuals that were collecting pollen from a feeder and in bees entering the hive loaded with pollen corbicules after recollection. For the first situation, we trained a group of bees to collect pure crushed multifloral bee-collected pollen from an artificial \u003cem\u003ead libitum\u003c/em\u003e feeder and marked them with acrylic paint of different colors to identify them during successive foraging visits. We touched the antennae of individual bees with a long stick (15 cm) embedded in sucrose sn. 40% or water shortly after the bee landed and started manipulating pollen. If bees extended their proboscis, we allowed them to ingest a drop of the solution (ca. 7 \u0026micro;l). The trials lasted 45 min, during which each bee made 3 to 5 trips. To measure acceptance inside the hive, we trained bees obtained from an observation hive to collect pollen from an artificial \u003cem\u003ead libitum\u003c/em\u003e feeder containing crushed bee-collected multifloral pollen. As before, we color-marked bees at the feeder to identify them at the foraging station and inside the hive. We measured sucrose and water acceptance immediately after focal bees enter the hive (before they unloaded their corbiculae in the hive cells; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). We removed the sides of the hive, which allowed us to access focal bees easily. Sucrose solution or water was randomly assigned, and each bee was presented with the same solution throughout all visits. When possible, we tested acceptance once per visit throughout all foraging visits or hive stays.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperiment 3. Testing learning and memory using pollen as co-reinforcement.\u003c/b\u003e We studied the differences in the acquisition and retention of olfactory memories during associative learning at the beginning and at the end of the foraging visit. Like nectar, bees also extend their proboscis when stimulated with pollen, however; this response is not stable, and bees often stop responding to pollen after a few events. Hence, we developed an alternative procedure in which we offer a simultaneous double reinforcement presenting sugar on the antennae and pollen on the tarsi of the first pair of legs. This procedure allows us to obtain stable PER responses throughout the successive training events. We olfactory conditioned bees by the presentation of the floral odor linalool (0.1 M, Sigma- Aldrich) as conditioned stimuli to both antennae complemented with sucrose-water solution (15%) as reward in the left antenna, and hand-collected kiwi pollen as reinforcement to the left first tarsi. Memories formed during 4 acquisition trials were then evaluated along 4 extinction trials that consisted in the presentation of the odor alone. We delivered the odor paired with sucrose\u0026thinsp;+\u0026thinsp;pollen (pollen paired procedure, PPP). To control possible sensitization produced by the effect of pollen stimulation on bees, we presented the odor paired to sucrose but not to pollen (pollen unpaired procedure, PUP). To control for mechanical stimulation, we presented the odor paired with sucrose\u0026thinsp;+\u0026thinsp;cellulose (cellulose paired procedure, CPP; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Despite being an inert compound, cellulose particles (which are similar in size to pollen grains) might provide a tactile stimulation that reinforces learning and memory of bees approaching the pollen source. Finally, bees that showed a spontaneous response (i.e. extending the proboscis in response to the first odor presentation) were excluded, as we cannot determine whether this is an innate response or if it indicates a prior (uncontrolled) odor- reward association.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOdor delivery\u003c/b\u003e. To present the CS, we used an olfactometer that sent a continuous clean air flow (50ml s-1) to the head and delivered the odor through a secondary air stream (6.25ml s-1) which was injected into the main airflow through a system of valves controlled by computer. A piece of filter paper (30x3mm) was impregnated with an aliquot of the odor (4\u0026micro;L) and placed inside a syringe connected to the secondary air stream. Each trial lasted 55s. The valve was programmed so that it released clean air during the first 20s, followed by the odor (6s), and a final exposure to clean air for the last 29s. The last 3s of the odor presentation overlapped with the sucrose\u0026thinsp;+\u0026thinsp;pollen (or cellulose) presentation in the paired procedures. During the unpaired procedures, we presented pollen or cellulose 5s after the odor\u0026thinsp;+\u0026thinsp;sucrose presentation. We measured the PER during the first 3s of the odor presentation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e For experiments 1 and 3 we used multiplicative generalized linear mixed models (GLMMs) assuming a Bernoulli distribution. When the PER occurred, we assigned values of 1, and when it did not, we assigned values of 0. In experiment 1, we analyzed the proportion of PER of restrained foragers considering forager type (a two-level factor corresponding to pollen and nectar foragers), foraging stage (a two-level factor corresponding to beginning and end), and sucrose concentration (continuous variable) as fixed effects. Individual bees and experimental days were considered as random effects. For experiment 2, we analyzed the PER proportion in response to sucrose and to water (a two-level factor) when they arrived at the feeder and when they entered to the hive (a two-level factor) by means of a binomial multiplicative generalized linear mixed model, using the \u0026ldquo;glmmTMB'' function of the \u0026lsquo;glmmTMB\u0026rsquo; package (Bates et al., 2015). Experimental days were considered as random effects. In experiment 3, we analyzed the proportion of PER of restrained pollen foragers during acquisition and extinction trials considering foraging stage (a two-level factor corresponding to beginning and end), treatment (a three-level factor corresponding to the reinforcement: paired pollen, unpaired pollen and cellulose), and trial (continuous variable) as fixed effects. Each bee was considered as random effects. We used the \u0026ldquo;glmer\u0026rdquo; function of the \u0026lsquo;lme4\u0026rsquo; package (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). For all the experiments, we conducted \u003cem\u003epost hoc\u003c/em\u003e contrasts on models to assess effects and significance between fixed factors using the \u0026ldquo;emmeans'' function of the \u0026lsquo;emmeans\u0026rsquo; package version 1.4 (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) with a significance level of 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch4\u003eAcknowledgements\u003c/h4\u003e\n\u003cp\u003eWe thank M.J. Corriale for her help with statistical analyses and W.M. Farina for the fruitful comments at the early stage of this paper. We also thank Wagner Chaves for their comments and suggestions.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eFunding\u003c/h4\u003e\n\u003cp\u003eThis study was partly supported by grants from Agencia Nacional de Promoción Cientıfica y Tecnológica (PICT_2017-2688) to A. Arenas and University of Buenos Aires (UBA-20020170100078BA).\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eData availability statement\u003c/h4\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBeshers, S. N. and Fewell, J. H. 2001. Models of division of labor in social insects. \u003cem\u003eAnnual review of entomology\u003c/em\u003e, 46, 413.\u003c/li\u003e\n \u003cli\u003eRobinson, G. E. 1992. 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The case against normal plots of effects. \u003cem\u003eJournal of Quality Technology\u003c/em\u003e. 47, 91-97. doi:10.1080/00224065.2015.11918111\u003c/li\u003e\n \u003cli\u003eLenth, R. 2019. emmeans: Estimated marginal means, aka least-squares means. https:// cran.r project.org/ web/packages/emmeans.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Foraging division of labor, Gustatory Sensitivity, Honeybee (Apis mellifera L), Foraging bout, Task specialization","lastPublishedDoi":"10.21203/rs.3.rs-2237679/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2237679/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDivision of labor is central to the ecological success of social insects. Among foragers of the honeybee specialization for collecting nectar or pollen correlates with their sensitivity to sucrose. So far, differences in gustatory perception have been mostly studied in bees returning to the hive, but not during foraging. Here, we showed that the phase of the foraging visit (i.e. beginning or end) interacts with foraging specialization (i.e. predisposition to collect pollen or nectar) to modulate sucrose and pollen sensitivity in foragers. In concordance with previous studies, pollen foragers presented higher sucrose responsiveness than nectar foragers at the end of the foraging visit. On the contrary, pollen foragers were less responsive than nectar foragers at the beginning of the visit. Consistently, free-flying foragers accepted less concentrated sucrose solution during pollen gathering than immediately after entering the hive. Pollen perception also changes throughout foraging, as pollen foragers captured at the beginning of the visit learned and retained memories better when they were conditioned with pollen\u0026thinsp;+\u0026thinsp;sucrose as reward than when we used sucrose alone. Altogether, our results support the idea that changes in foragers' perception throughout the foraging visit contributes to task specialization.\u003c/p\u003e","manuscriptTitle":"Changes in resource perception throughout the foraging visit contribute to task specialization in the honeybee Apis mellifera","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-14 16:10:12","doi":"10.21203/rs.3.rs-2237679/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-03-07T10:04:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-02-27T15:32:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24e23294-756a-492f-b99b-d5e4d6e03b41","date":"2023-02-24T10:18:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-02-24T02:56:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-12-28T07:43:22+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-11-11T12:04:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-11-11T11:58:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-11-04T10:33:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"15c03f3c-7bff-48a6-8fe8-4fdf4fb90b18","owner":[],"postedDate":"November 14th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":16898020,"name":"Biological sciences/Zoology"},{"id":16898021,"name":"Biological sciences/Zoology/Animal behaviour"},{"id":16898022,"name":"Biological sciences/Zoology/Animal physiology"},{"id":16898025,"name":"Biological sciences/Zoology/Entomology"},{"id":16898027,"name":"Biological sciences/Neuroscience"},{"id":16898028,"name":"Biological sciences/Neuroscience/Learning and memory"},{"id":16898029,"name":"Biological sciences/Neuroscience/Olfactory system"},{"id":16898030,"name":"Biological sciences/Neuroscience/Social behaviour"}],"tags":[],"updatedAt":"2023-10-16T21:07:28+00:00","versionOfRecord":{"articleIdentity":"rs-2237679","link":"https://doi.org/10.1038/s41598-023-35163-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2023-05-19 20:53:10","publishedOnDateReadable":"May 19th, 2023"},"versionCreatedAt":"2022-11-14 16:10:12","video":"","vorDoi":"10.1038/s41598-023-35163-y","vorDoiUrl":"https://doi.org/10.1038/s41598-023-35163-y","workflowStages":[]},"version":"v1","identity":"rs-2237679","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2237679","identity":"rs-2237679","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}