Learning and memory in workers reared by nutritionally stressed honey bee (Apis mellifera L.) colonies

Physiology & Behavior 95 (2008) 609–616

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Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b

Learning and memory in workers reared by nutritionally stressed honey bee (Apis mellifera L.) colonies Heather R. Mattila ⁎, Brian H. Smith 1 Department of Entomology, Ohio State University, Columbus, Ohio, USA

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Article history: Received 21 December 2007 Received in revised form 20 July 2008 Accepted 4 August 2008 Keywords: Behavioral assays Conditioned learning Honey bee workers Latent inhibition Memory Nutritional stress PER reflex Pollen diet Sucrose-response thresholds

a b s t r a c t Chronic nutritional stress can have a negative impact on an individual's learning ability and memory. However, in social animals that share food among group members, such as the honey bee (Apis mellifera L.), it is unknown whether group-level nutritional stress is manifested in the learning performance of individuals. Accordingly, we examined learning and memory in honey bee workers reared by colonies exposed to varying degrees of long-term pollen stress. Pollen provides honey bee workers with almost all of the proteins, lipids, vitamins, and minerals that they require as larvae and adults. Colonies were created that were either chronically pollen poor or pollen rich, or were intermediate in pollen supply; treatments altered colonies' pollen stores and brood-rearing capacity. Workers from these colonies were put through a series of olfactoryconditioning assays using proboscis-extension response (PER). PER thresholds were determined, then workers learned in olfactory-conditioning trials to associate two floral odors (one novel and the other presented previously without reward) with stimulation with sucrose and a sucrose reward. The strength of the memory that was formed for the odor/sucrose association was tested after olfactory-conditioning assays ended. Colony-level nutritional status had no effect on worker learning or memory (response threshold of workers to sucrose, acquisition of the odor/sucrose association, occurrence of latent inhibition, or memory retention over 72 h). We conclude that potential effects of chronic, colony-wide nutrient deprivation on learning and memory are not found in workers, probably because colonies use brood-rearing capacity to buffer nutrient stress at the level of the individual. © 2008 Elsevier Inc. All rights reserved.

1. Introduction Chronic nutritional stress, particularly early in life, has been shown to induce learning and memory deficiencies in numerous vertebrate models (e.g., mice, rats, songbirds, sugar gliders, and humans [8,10,15,20,21,38,40,41,46,47,57,59–61]). Most studies enforce diet restrictions under laboratory conditions, although analogous scenarios of nutritional stress arise in nature when parents distribute food among their offspring or when constrained resources are otherwise shared by groups of individuals. Nevertheless, it is difficult to know the extent to which artificially induced diet restrictions emulate the effects of nutritional stress under natural conditions. For example, artificial manipulation of food availability during early growth affects neural development, song acquisition, and repertoire size in songbirds [40,41,57], but these effects can largely disappear when parents are allowed to provision their brood naturally, even when food is limited,

⁎ Corresponding author. Current address: Department of Neurobiology and Behavior, Cornell University, Ithaca, New York 14853, USA. Tel.: +1 607 254 4377; fax: +1 607 254 1303. E-mail address: [email protected] (H.R. Mattila). 1 Current address: School of Life Sciences, Arizona State University, Phoenix, Arizona 85284, USA. 0031-9384/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2008.08.003

brood size is large, and offspring show other signs of developmental stress (e.g., reduced weight and size; [17]). It is possible that if control over the distribution of limited resources among a group is retained by its members, then the collective is better positioned to buffer the impact of group-level stress on critical attributes of learning in its individuals. Honey bee (Apis mellifera L.) colonies present an excellent opportunity to investigate whether group-level nutritional stress is manifested in the learning ability of individuals. Learning is an unquestionably vital adaptation for honey bees during their lifetimes, for such activities as navigating the environment, foraging on various flowers, or orienting to the nest entrance. Furthermore, honey bee colonies are under strong selective pressure to maintain nest homeostasis by buffering fluctuations in both the availability of food in the environment and reserves stored in the nest [54]. In temperate climates, colonies have less than eight weeks annually during which they can stockpile food reserves to fuel nest activities over the remainder of the year [34,55]. Consequently, there are extended “lean” periods during which colony resources are constrained and food reserves dwindle as they are used to support the nutritional demands of the larval and adult population. As the primary dietary source of proteins, lipids, vitamins, and minerals, pollen provides most of the nutrients required by adult

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honey bees and developing brood for growth and function [24]. When pollen deprivation is enforced, malnutrition can result in undersized workers [2,16,26], protein loss [23], shortened lifespans and reduced nursing capacity [35], and an impaired ability to overcome signs of disease [4,50,62]. If workers are allowed to control the distribution of resources among individuals, then colonies are better able to compensate for fluctuations in pollen supply by tailoring broodrearing intensity to resource availability [32]. Nonetheless, the effects of colony-level pollen stress are often revealed in features of worker function [25,33,35,53,63], which demonstrates that the nutritional state of the colony has the potential to affect worker traits under natural conditions. Despite its influence on worker physiology and function, the effects of a colony's nutritional status on the learning ability of its workers have never been examined. We explored the nutrition-learning relationship by investigating the impact of long-term, colony-level pollen stress on the learning performance of workers. A potent type of learning in honey bees is olfactory-associative (or conditioned) learning, where workers learn to associate odors with stimuli that precede rewards [22,36,37]. The rapid acquisition of the learned association enables workers to gather useful information about signals that predict rewards, and long-term memories lasting from days to a lifetime are typically formed in the process [22,37]. Effects of nutritional state on brain development, learning, and memory have received considerable attention in vertebrate models [10,38,41,61], although the same questions have been largely ignored in studies of invertebrates. A singular example has been documented in Drosophila, where it was found that flies reared on diets deficient in proteins, fats and minerals – the same nutrients that are derived almost exclusively from pollen by honey bees – exhibited impaired associative learning abilities and memory as adults [19,66]. We determined whether similar learning and memory deficiencies are found in honey bees reared in environments that varied in their degree of long-term, colony-level pollen stress. It is an especially relevant question given the widespread use of the honey bee as a model for learning studies and the dearth of knowledge regarding the potential effects of the nutritional state of colonies on the learning performance of workers. Our studies were conducted using an olfactory-conditioning paradigm that employs the proboscis extension response (PER) reflex of workers [7,22]. Honey bees respond to sucrose stimulation (an unconditioned stimulus, US) of sucrose receptors on the antennae by reflexively extending their mouthparts. This response can be released by an odor-conditioned stimulus (CS) if presentation of the odor is immediately followed by the US; an increase in probability of response to the odor CS occurs after only one or a few pairings with the US. PER is a powerful tool for assaying learning ability in honey bees under controlled conditions. It is a robust test of performance that incorporates many of the features of associative learning that are observed in animals under natural conditions [6,39]. Furthermore, it is sensitive to learning impairment induced by environmental stressors such as exposure to pesticides [11,12], mite infestation [29], or changes in nest temperature during pupation [27,58]. In order to increase sensitivity to stress-related performance deficits, we used PER in four different behavioral measures of associative and non-associative learning. Our protocol examined workers for: 1) their response threshold to sucrose [42], 2) the occurrence of latent inhibition [9], 3) the rate of acquisition of conditioned PER to two floral odors (one familiar, one novel), and 4) memory retention of the learned association. Changes in sucrose response thresholds (the lowest concentration of sucrose solution that elicits PER) are correlated with changes in associative learning [51] and can reflect differences in the nutritional status of workers (i.e., access to sucrose, unpublished data [44]). Therefore, differences in response thresholds of workers could suggest differences in the perceived nutritional state of colonies and reflect simultaneous changes in learning. Latent

inhibition is a phenomenon whereby subjects demonstrate a slower rate of conditioning to a CS when they have been pre-exposed to that CS without reward. Latent inhibition has been documented in many animals, including the honey bee [1], and its occurrence provides a measure of a subject's ability to disregard stimuli that are meaningless, such as a forager ignoring odors that do not predict food rewards. Finally, rate of acquisition and memory of the learned CS/US association were investigated as measures of the ability of workers to learn and retain conditioned PER, and are commonly cited measures for comparison of associative learning in honey bees [22]. Collectively, these behavioral assays provide a robust test of the effects of colonylevel nutritional state on learning and memory of worker honey bees. 2. Materials and methods 2.1. Establishing colonies The study was conducted at the Rothenbuhler Honey Bee Laboratory on the Ohio State University campus in Columbus, Ohio, from July to August 2004. All research was completed during a seasonal resource dearth that was typical of the region. In mid-July, nine colonies were assembled using bees and brood-filled and foodfilled comb from previously established colonies. Each colony was given nine frames of comb: two frames filled with honey, five frames of brood and pollen and two empty frames. Colonies were provided with similar amounts of brood (see measurements of capped-pupae brood taken 21 July) and similar numbers of workers (approximately seven frames covered with bees). Brood frames that contained pollen were distributed differentially among the colonies so that some colonies had a lot of pollen, others had very little, and the remainder had intermediate amounts of pollen (see Section 2.2 for further details). Sister queens of Carniolan descent were introduced to each colony two days after the colonies were established. The queens were reared at the lab in 2004 and were naturally mated within a closed breeding population (the OSU campus apiary is isolated in the center of a large urban area). 2.2. Manipulating colony-level nutritional status Colonies were placed in one of three treatment groups based on the amount of pollen provided to them initially and on access to external pollen resources thereafter (n = 3 colonies per treatment group). 2.2.1. Pollen-poor colonies Colonies that were established initially with small amounts of stored pollen (mean 140 ± 73 cm2 of pollen-filled comb per colony). Their reserves were similar to stores in colonies where worker performance and activities were affected by constrained pollen resources [32,33]. These colonies were also fitted with pollen traps that removed pollen pellets from foragers when they returned to colonies. The traps were emptied every two to three days and the pollen was frozen at -20 °C until it was used to supplement the pollen supply in the pollen-rich colonies (see Section 2.2.2). Over two months, an average of 2.2 kg of pollen was removed from each colony by the traps. 2.2.2. Pollen-rich colonies Colonies that were established initially with large amounts of pollen (1420 ± 220 cm2 of pollen-filled comb per colony) were also supplemented with pollen patties ad libitum throughout the study. Patties (500 g) were made by mixing (w/w) two parts pollen (collected from pollen-stressed colonies) with one part sugar syrup (67% granulated sugar in water, v/v) and then pressing the mixture between two sheets of waxed paper. Patties were placed near the

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brood area and were replaced as they were consumed. Each pollenrich colony received approximately 5 kg of additional pollen over the course of the study. 2.2.3. Colonies with intermediate pollen supply Colonies provided initially with intermediate amounts of pollen (mean 650 ± 168 cm2 of pollen-filled comb per colony) were permitted to collect, store, and use pollen without interference for the duration of the study. 2.3. Workers used for PER assays From each source colony, three cohorts of newly eclosed workers were collected and then marked with paint so that workers of known ages could be used in the PER assays; workers were marked as they emerged from capped-pupae cells on 6, 14, and 20 August. Emerging workers were obtained from brood frames that were taken from each source colony and placed overnight in an incubator (34 °C, 65% relative humidity). Once marked, workers were returned to their source colonies, where they remained until they were collected again for the assays. Pollen treatments were implemented in mid-July, thus all focal workers were reared and lived under the conditions of colony-level pollen stress that were created in their colonies. The size of pollen stores in colonies, a reflection of colony-level nutritional status, were no longer significantly different across treatment groups in only the final days that the last cohort of workers was assayed (Fig. 1). For each cohort of paint-marked workers in a colony, four marked workers were collected daily from the brood frames over five consecutive days, when marked workers were 8–12 days of age, to be tested in the PER assays. This sampling regimen was repeated for all three cohorts. Each worker was collected from her colony in a ventilated glass vial, subdued on ice, and then harnessed in a small tube [22]. Workers were mounted with their heads sticking out of the tube so that their heads and mouthparts could move freely for the PER assays, but their appendages, thoraxes, and abdomens were restrained. After workers recovered for 0.5 h, they were put through a sequential series of tests: a PER response-threshold assay, latentinhibition training, a PER olfactory-conditioning assay (all in the same day), and a memory-retention assay (over a subsequent 72-h period after initial assays ended). 2.4. Assaying PER-response thresholds to sucrose Workers were assayed with a series of sucrose solutions that increased in concentration (0.1, 0.3, 1, 3, 10, and 30% w/w sucrose

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solution in water) to determine the lowest concentration of sucrose that would elicit PER [42]. Stimulation with sucrose solution was alternated with water to reduce potential sensitization and habituation that can occur with high sucrose concentrations and repeated testing with sucrose [43]. Harnessed workers were lined up and tested sequentially for PER to the same concentration of sucrose, a droplet of which was touched briefly to each individual's antenna. A trial with a single sucrose solution was executed rapidly before workers were tested with water and then with the next sucrose solution in a subsequent trial. The inter-trial interval (ITI = the length of time between tests on the same worker during which all the other workers were assayed) did not exceed 3 min. Subjects were always tested in the same order to keep the ITI constant between each trial. Workers were scored as having a positive PER if they extended their proboscis fully (beyond a line extending between the tip of the mandibles) when their antenna was touched with each solution. No PER or partial movements of the mouthparts were recorded as a negative response. PER response-threshold scores were determined for each individual by summing the number of positive PERs given by that worker to the six sucrose solutions [44]. Workers tend to respond consistently with PER once the response threshold is reached ([43]; HRM pers. obs.). Thus, a high PER response-threshold score meant that workers responded to most sucrose solutions, including low concentrations, and had a low response threshold to sucrose. Conversely, workers with a low score were more likely to respond to only the most concentrated sucrose solutions and therefore had a high response threshold. 2.5. Latent-inhibition training Of the four workers collected daily from each colony for responsethreshold assays, two were randomly selected to undergo subsequent latent-inhibition training [9] and olfactory-conditioning assays (workers not demonstrating PER in the previous assay were excluded if responding workers were available). Eighteen workers were trained per day (three treatments × three colonies per treatment × two workers per colony). Before workers were conditioned to associate two odors (CSs) with sucrose (US), they were first exposed to one of the odors (odor A) over 40 trials without presentation of the US or reinforcement with reward. By continually exposing workers to odor A without reinforcement, we expected in the next assay that workers would acquire the CS/US association more slowly once it was reinforced with a sucrose reward compared to acquiring the association with novel odor B. Odor A was alternated daily between the two floral odors 2.0 M 1-hexanol and 2-octanone (the other was assigned as odor B by default). Each harnessed worker was positioned 2 cm from the end of a 1-cc glass tuberculine syringe loaded with odor-soaked filter paper (5 μL of odor A). The syringe was attached to an air pump via a valve that shunted odor-filled air towards each worker for 4 s every 5 min (ITI). This procedure was repeated 40 times over a period of 3 h 20 min for each worker; all 18 workers were trained simultaneously each day. Each syringe was replaced with a fresh one after six odor deliveries. A ventilation system was in place behind the workers to ensure that the odor was removed quickly from the area once it was delivered. Olfactory-conditioning assays began 10 min after latent-inhibition training ended. 2.6. Assaying acquisition of conditioned PER

Fig. 1. Mean area of pollen-filled comb per colony (±s.e) estimated over the course of the study for colonies experiencing varying long-term pollen supply. Letters indicate significant differences across survey dates in pollen stored by colonies.

During the olfactory-conditioning assays, workers learned that the presentation of CS odors predicted reward with sucrose. Odor A (familiar from latent-inhibition training) and odor B (novel) were presented to each subject in a pseudo-randomized manner over 12 trials (ABBABAABABBA). Each worker had a 5-min ITI between trials, during which time the other 17 workers were assayed sequentially.

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Similar to the protocol for latent-inhibition training, harnessed workers were positioned at the end of a syringe containing odorsoaked filter paper and a stream of air was directed through the syringe and toward the worker for 6 s when a valve system was triggered. After 4 s of exposure to odor alone, the subject's antenna was touched briefly with a 0.2 mL droplet of 30% w/w sucrose solution. When the subject responded with PER, it was rewarded with a droplet of the sucrose solution. Care was taken to ensure that the sucrose reward was provided to the worker before the odor-filled air stopped flowing. A ventilation system behind the assay apparatus removed the odor from the area after it was delivered to the worker. Subjects showing PER to odor alone (i.e., during the 4 s before antennal stimulation with the sucrose) were scored as providing a positive conditioned response. The rate of acquisition of the CS/US association was determined over six trials with each odor. Conditioned-PER scores for each odor, similar to those described for response threshold, were calculated for each worker over six trials [9]. At the end of each day, when response-threshold assays, latentinhibition training, and olfactory-conditioning assays were complete, workers were fed to satiety with 1.5 M sucrose solution and then transferred to a humidified container so that surviving workers could be assayed for retention of the memory of the learned CS/US association over the next 72-h period. 2.7. Assaying memory retention Workers were evaluated for conditioned PER to odors A and B at 24, 48, and 72 h after they first learned the CS/US association. Both odors were presented to workers at 24-h intervals as described for the olfactory-conditioning assays, but this time without reward. PER was scored as either positive or negative. The order in which odors were presented was alternated daily. After responses to both odors were assayed, workers were again fed to satiety with 1.5 M sucrose and returned to the humidified container. Memory retention was examined by determining the percentage of workers that showed a positive PER over time to both odors. Further, PER memory-retention scores, similar to those calculated for response-threshold and olfactory-conditioning assays, were determined for each worker. Only workers that survived for the entire 72-h period were included in the analyses (73% of those that were assayed initially). 2.8. Monitoring brood production and pollen stores in colonies The area of comb occupied by capped-pupae cells was monitored in colonies throughout the study to estimate changes in brood rearing as a result of colony-level pollen status. Pollen stores were also quantified as an indicator of the difference in pollen availability created among treatments. Estimates of both variables were made in each colony at four different dates over the course of the study (21 July and 6, 19, and 29 August) by placing a metal grid (2.5 × 2.5 cm squares) over each side of every frame in a colony and counting the number of squares that were filled with either capped brood or pollen stored in comb. Partially filled squares were estimated as half of a square and were added to the total. The number of squares that were filled with capped brood or pollen was summed for each colony and converted to an area estimate (cm2). These data were compared among treatments and across time. 2.9. Statistical analyses In total, 540 workers were assayed for their PER responsethresholds to sucrose, 270 workers were trained in the olfactoryconditioning assays and 198 workers were tested for their memory of the CS/US association. All statistical analyses were conducted with SAS Version 8.2 (SAS Institute Inc., Cary, USA). Two-, three- or four-way

analyses of variance (ANOVAs) for a completely randomized design were performed depending on the assay (Proc GLM or Proc Mixed if repeated measures was a factor). Potential factors included pollen treatment (pollen-poor, pollen-rich, or intermediately supplied colonies), cohort-eclosion date (6, 14, or 20 August), worker age (812 days of age), and odor type (familiar odor A or novel odor B). In instances where values were calculated for multiple individuals from each colony (e.g., PER scores), a colony mean was determined and these were examined with an ANOVA. Percent data were arcsine transformed prior to analysis [56]. Means are reported with standard errors (s.e.). Means for significant effects were separated using Tukey's studentized range test. The significance level for all tests was set at α = 0.05. 3. Results 3.1. Effects of treatment manipulations on pollen stores The amount of pollen stored in comb by colonies was affected by the implementation of treatments to alter the pollen status of colonies (F[2, 6] = 10.7; p = 0.01) and differed over the dates that colonies were surveyed (F[3, 18] = 4.6; p = 0.01; treatment × survey date interaction: F[6, 18] = 4.1; p = 0.01; Fig. 1). At the beginning of the study, there were large disparities across treatment groups in the amount of pollen that was stored in colonies (21 July survey; Fig. 1). Differences among pollen-poor, pollen-rich, and intermediately supplied colonies in mean pollen stored per colony persisted over most survey dates, but the size of the colonies' pollen stores converged by the end of the study (Fig. 1). 3.2. Effects of nutritional state on PER-response thresholds to sucrose Response thresholds of workers to sucrose were not influenced by the degree of pollen stress in colonies (F[2, 90] = 0.6; p = 0.54). Mean PER response-threshold scores were similar across treatment groups and ranged from 2.9 ± 0.19 in the pollen-poor and intermediately supplied colonies to 3.2 ± 0.19 in the pollen-rich colonies. These scores mean that, on average, the lowest concentration of sucrose that elicited PER in workers was 1% (w/w) sucrose. Response thresholds were sensitive to both worker age (F[4, 90] = 2.7; p = 0.04) and eclosion date (F[2, 90] = 7.9; p = 0.001). Workers that eclosed on 6 August had higher PER response-threshold scores than workers that eclosed on 20 August (mean scores of 2.3 ± 0.19 versus 3.3 ± 0.19) and workers that were 9 days of age had higher PER response-threshold scores than workers that were 11 days of age (mean scores of 2.5 ± 0.24 versus 3.4 ± 0.24); significant differences were not found among other treatment mean combinations. 3.3. Effects of nutritional state on acquisition of conditioned PER The degree of pollen stress in colonies had no effect on the ability of workers to acquire the association between the odors (CSs) and sucrose (US) (Fig. 2; comparison of conditioned-PER scores; F[2, 180] = 1.1; p = 0.35). Most workers learned the CS/US association quickly; after only one previous pairing of each odor with sucrose, over 50% of workers responded with a positive PER to either odor alone, across all treatment groups, and regardless of the odor used (Fig. 2). By the third trial, over 60% of workers had demonstrated acquisition of conditioned PER to both odors; the percentage of workers that showed conditioned PER to the odors leveled off in subsequent trials and never fell below 60% for any of the treatment groups (Fig. 2). Similar to PER response-thresholds scores, conditioned-PER scores that gauged acquisition were influenced by the eclosion date of workers (F[2, 180] = 6.7; p = 0.002) and worker age (F[4, 180] = 5.4; p b 0.001). Scores were lower for workers that eclosed on 6 August

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3.5. Effects of nutritional state on memory retention Although the percentage of workers that showed a positive PER generally decreased at each 24-h interval after olfactory-conditioning assays ended (effect of time: F[2, 72] = 19.5; p b 0.001; Fig. 3), memory retention of workers was not influenced by the pollen status of their colonies (F[2, 36] = 1.1; p = 0.35; time × treatment interaction: F[4, 72] = 0.1; p = 0.97; Fig. 3) or by the odor that was assayed (F[1, 36] = 0.1; p = 0.71; Fig. 3). Similarly, conditioned-PER memory-retention scores showed no effect of colony-level nutritional state on worker memory (F[2, 180] = 0.1; p = 0.89) and no difference in response between odors (F[1, 180] = 0.5; p = 0.48). 3.6. Effects of nutritional state on colony-level investment in brood Colony-level nutritional state affected investment in brood rearing throughout the study (F[2, 6] = 21.4; p b 0.01; effect of survey date: F[3, 18] = 42.5; p b 0.001; treatment × survey date interaction: F[6, 18] = 6.3; p b 0.01; Fig. 4). Colonies were provided with similar amounts of pupal brood when the study was initiated according to the 21 July survey, but in subsequent surveys, pollen-rich colonies showed increased investment in brood rearing relative to pollen-poor colonies or colonies that were supplied with intermediate amounts of pollen (Fig. 4).

Fig. 2. Mean percentage of workers (± s.e.) showing conditioned PER to the presentation of (i) familiar odor A (presented previously without reward) and (ii) novel odor B; odors (CS) were paired with sucrose (US) in an olfactory-conditioning assay. Workers were reared in colonies that varied in their long-term supplies of pollen.

compared to 14 or 20 August (2.7 ± 0.16 versus 3.5 ± 0.16 and 3.4 ± 0.16) and lower for 9 and 10 day-old workers compared to 8 and 11 day-old workers (2.8 ± 0.20 and 2.7 ± 0.20 versus 3.8 ± 0.20 and 3.6 ± 0.20). 3.4. Effects of nutritional state on the occurrence of latent inhibition There was a significant difference in the response of workers to familiar odor A and novel odor B the first time that they were presented to workers. During the first trial, less than 1% of workers showed spontaneous PER to the previously unrewarded and familiar odor A before initial presentation of the US (i.e., they showed almost no likelihood of extending their proboscis when presented with odor A after repeated pre-exposure without sucrose stimulus or reward; Fig. 2A), whereas 15–23% of workers gave a spontaneous PER to novel odor B the first time it was presented to them (Fig. 2B; effect of odor in the first trial: F[1, 180] = 45.8; p b 0.001). This difference in response to each odor, which results from unrewarded pre-exposure to odor A, was not affected by the amount of pollen available in the source colonies (Fig. 2; F[2, 180] = 1.6; p = 0.21; effect of treatment × odor interaction: F[2, 180] = 1.5; p = 0.23). Differences in the response of workers to the odors were no longer detected after the first trial (Fig. 2; effect of odor in the second trial: F[1, 180] = 3.4; p = 0.06; third trial: F[1, 180] = 0.2; p = 0.64; fourth trial: F[1, 180] = 0.2; p = 0.66; fifth trial: F[1, 180] = 0.001; p = 0.98; sixth trial: F[1, 180] = 0.4; p = 0.51; no significant treatment × odor effects), which suggests that the effects of latent inhibition were overcome after the first pairing of familiar odor A with sucrose.

Fig. 3. Mean percentage of workers (± s.e.) showing conditioned PER when presented with (i) odor A and (ii) odor B (both without reward) at 24, 48, and 72 h after olfactoryconditioning assays ended. Workers were reared in colonies that varied in their longterm supplies of pollen. Letters indicate significant differences between the mean percentages of workers that responded positively at each 24-h interval (pooled across odors and treatment groups because they were not significant effects).

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Fig. 4. Mean area of capped-pupae-filled comb per colony (± s.e.) estimated over the course of the study for colonies that varied in their long-term supplies of pollen. Letters indicate significant differences across survey dates in the mean area of comb occupied by pupal brood in colonies.

These latter two groups produced indistinguishable amounts of brood over the course of the study (Fig. 4). Worker production peaked on the 6 August survey in all treatment groups before returning to lower levels (Fig. 4). 4. Discussion The effects of malnutrition on learning and memory, often documented in vertebrates [8,10,15,20,21,38,40,41,46,47,57,59–61] and studied in only one comparable insect model, Drosophila [19,66], seem unlikely to be found in workers reared by nutritionally stressed honey bee colonies. Our tests of associative learning using conditioned PER did not detect any effects of colony-level pollen status on the performance of workers, in spite of the use of several means for evaluating behavior and the significant effects that treatments had on both brood rearing and pollen storage. The ability of workers to acquire the association between floral odors and sucrose rewards was not disrupted even when colony-level pollen reduction was prolonged and spanned from larval development to adulthood. Performance of learning tasks was enhanced in Drosophila reared on diets that were enriched with the same nutrients provided to honey bees by pollen [66], but similar effects were not seen for associativelearning ability of workers reared in pollen-rich colonies in this study. Further, workers from both pollen-rich and pollen-poor colonies demonstrated similar memory retention, comparable response thresholds to sucrose, and the same degree of latent inhibition. The interesting question to ask at present is: why do such profound colony-level differences in nutritional state have little or no effect on the learning performance of individual workers? When colony members are permitted to allocate resources among workers and brood without interference, they show a remarkable ability to compensate for food shortages. Pollen-deprived colonies accomplish this adjustment by reducing investment in brood rearing [32] and by increasing pollen stores, probably through increased pollen-foraging effort [14,31,64], thereby minimizing potential impacts of dearths on the performance of individual workers. Adjustments to alleviate colony-level pollen stress happen fast in honey bees. In our study, colonies converged at similar levels of stored pollen by the end of the study, even after we created substantial differences in the size of pollen reserves initially and hampered the ability of colonies to recover these resources from the environment. A tendency for colonies to restore their pollen reserve to an equilibrium set-point after significant experimental manipulation has been documented previously [14]. This scenario contrasts with the discovery of learning

deficits in malnourished Drosophila, where nutrient constraints were imposed at the level of the individual [66] and adjustments through a group-provisioning strategy were not an option. We are not able to determine whether similar effects on learning would be found if diet stress was enforced for individual honey bee workers; the well-tuned response of colonies to fluctuations in resource availability reduces the likelihood of finding significant effects of colony-level nutritional status on worker cognition. We believe that PER assays are sensitive enough to detect potential changes in worker learning caused by colony-level nutritional status, had they been present. Similar protocols have found changes in response thresholds and associative learning caused by environmental stress [11,12,27,29,44 (unpubl. data), 58], and all of our assays revealed significant differences in conditioned PER for workers that were only days apart in age or eclosion date. These results are in line with previous studies that have documented the effect of age and season on associative learning using PER assays [5,11,30,45,48,49,51]. The fact that our assays detected differences in conditioned PER of workers reared just one week apart (among cohorts) or with only a one-day difference in age (within cohorts) indicates the sensitive nature of conditioned PER to subtle changes in the environment and worker physiology. Furthermore, we predict that effects of malnutrition on worker learning would be found if control of food distribution was stripped from colony members and nutritional stress was imposed at the level of the individual. This prediction is based on knowledge of the effects of enforced starvation on worker physiology [23,26,35] and the impact of other environmental stressors on neurodevelopment and associative learning in honey bees [11,12,18,27,29]. Effects on neurodevelopment of enforced, chronic nutritional stress during early life are especially pertinent in the honey bee because the mushroom bodies, brain structures that are involved in olfactory learning and memory in adult workers, particularly those of foraging age [65], are laid down during larval and pupal development [13] and disruption of this process can lead to learning deficits in adult honey bees [28,52] and other insect models [3]. It is possible that there may be effects of colony-level nutritional stress on the learning ability and memory of workers, but that these effects were not reflected in conditioned PER of workers in these assays. In honey bees, olfactory-associative learning is a fundamental component of worker learning that is critically important for optimizing foraging success, and harnessed workers often learn odors that predict food rewards after only a single conditioning trial ([22] and this study). Consequently, the neural structures that support olfactory-associative learning may be highly conserved and resistant to disruptions related to poor nutrition during development and adulthood, although dysfunctional neurodevelopment has been identified previously as a culprit for stress-induced learning impairments in honey bees [58] and could be expected in this case as well. Even if olfactory-associative learning was not affected by pollen stress, other categories of learning may be susceptible to injury caused by colony-level food shortages. Inferences regarding global learning deficiencies in workers (beyond olfactory-conditioned PER) caused by colony-level malnutrition could be made by evaluating the successful performance of tasks that depend on a combination of learning skills. For example, differences between workers reared in pollen-poor colonies and workers reared in colonies with adequate or abundant food supplies may be revealed in the amount and quality of food that workers collect as foragers, or in their relative ability to convey information about food resources through waggle dances. This study is the first to explore the effects of the nutritional status of colonies on the learning ability of worker honey bees. The absence of an effect within an olfactory-conditioning assay is important to document because it is a method that is commonly used to evaluate the cognitive performance of workers in studies of honey bee learning [27 and references cited therein]. Previous work has shown that changes in nest temperatures during brood development have

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significant effects on the learning ability and short-term memory of adult workers in olfactory-conditioning assays [27,58], and that these learning deficiencies are linked directly to difficulties in dance communication as foragers [58] and disruption of normal brain development [18]. This knowledge has motivated researchers to monitor closely the temperatures at which they incubate developing brood that are destined to be used in learning trials as adults. Further studies are required to explore other effects of stress on learning in honey bees, but our results imply that honey bee colonies have a significant capacity to buffer the impact of colony-level nutritional stress such that the learning performance of workers is protected. Moreover, these results have important implications for understanding how a social animal such as the honey bee allocates collective resources among colony members during times of stress. Acknowledgments We would like to thank the staff and students who were part of the Rothenbuhler Honey Bee Laboratory (OSU) when the study was conducted; we especially appreciated technical advice from S. Cobey and S. Kottcamp. HRM is also grateful to the members of the Department of Environmental Biology at the University of Guelph, her home institution when she visited OSU. This manuscript benefited from critical readings by D. Gibo, G. Otis, J. Schmidt, and G. Umphrey. Funding for HRM's travel to Ohio was provided by a Graduate Research Travel Grant from the Entomological Society of Canada. The research was supported by a Canada Graduate Scholarship and a Postdoctoral Fellowship from the Natural Sciences and Engineering Research Council of Canada (HRM) and a grant from National Institutes of Health National Center for Research Resources (grant no. RR014166 to BHS). References [1] Abramson CI, Bitterman ME. Latent inhibition in honeybees. Anim Learn Behav 1986;14:184–9. [2] Alpotov WW. Biometrical studies on variation and races of the honey bee (Apis mellifera L.). Q Rev Biol 1929;4:1–58. [3] Belle JS de, Heisenberg M. Associative odor learning in Drosophila abolished by chemical ablation of mushroom bodies. Science 1994;263:692–5. [4] Beutler R, Opfinger E. Pollenernährung und Nosemabefall der Honigbiene (Apis mellifica). Z Ver Physiol 1949;32:383–421. [5] Bhagavan S, Benatar S, Cobey S, Smith BH. Effect of genotype but not age or caste on olfactory learning performance in the honey bee, Apis mellifera. Anim Behav 1994;48:1357–69. [6] Bitterman ME. Comparative analysis of learning in honeybees. Anim Learn Behav 1996;24:123–41. [7] Bitterman ME, Menzel R, Fietz A, Schäfer S. Classical conditioning of proboscis extension in honeybees (Apis mellifera). J Comp Psychol 1983;97:107–19. [8] Bush M, Leathwood PD. Effect of different regimens of early malnutrition on behavioural development and adult avoidance learning in Swiss white mice. Brit J Nutr 1975;33:373–85. [9] Chandra SBC, Hosler JS, Smith BH. Heritable variation for latent inhibition and its correlation with reversal learning in honeybees (Apis mellifera). J Comp Psychol 2000;114:86–97. [10] Dauncey MJ, Bicknell RJ. Nutrition and neurodevelopment: mechanisms of developmental dysfunction and disease in later life. Nutr Res Rev 1999;12:231–53. [11] Decourtye A, Lacassie E, Pham-Delègue MH. Learning performances of honeybees (Apis mellifera L.) are differentially affected by imidacloprid according to the season. Pest Manag Sci 2003;59:269–78. [12] Decourtye A, Devillers J, Genecque E, Le Menach K, Budzinski H, Cluzeau S, et al. Comparative sublethal toxicity of nine pesticides on olfactory learning performances of the honeybee Apis mellifera. Arch Environ Con Tox 2005;48:242–50. [13] Farris SM, Robinson GE, Davis RL, Fahrbach SE. Larval and pupal development of the mushroom bodies in the honey bee, Apis mellifera. J Comp Neurol 1999;414: 97–113. [14] Fewell JH, Winston ML. Colony state and regulation of pollen foraging in the honey bee, Apis mellifera L. Behav Ecol Sociobiol 1992;30:387–93. [15] Fisher MO, Nager RG, Monaghan P. Compensatory growth impairs adult cognitive performance. PLOS Biol 2006;4:1462–6. [16] Fyg, W. Normal and abnormal development in the honeybee. Bee World 1959, 40:57–66, 85–96. [17] Gil. D, Naguib M, Riebel K, Rutstein A, Gahr M. Early condition, song learning, and the volume of song brain nuclei in the zebra finch (Taeniopygia guttata). J Neurobiol 2006;66:1602–12.

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