The effect of foraging specialization on various

Behav Ecol Sociobiol (2009) 64:135–148 DOI 10.1007/s00265-009-0829-z

ORIGINAL PAPER

The effect of foraging specialization on various learning tasks in the honey bee (Apis mellifera) Tamar Drezner-Levy & Brian H. Smith & Sharoni Shafir

Received: 5 January 2009 / Revised: 1 July 2009 / Accepted: 2 July 2009 / Published online: 15 July 2009 # Springer-Verlag 2009

Abstract Honey bee foragers may collect nectar, pollen, water, or propolis, and their foraging specialization has been associated with several behavioral traits. By conditioning of the proboscis extension response (PER), we compared the performance of foragers that collected nectar, pollen, both nectar and pollen, or water in several learning and choice assays. Foragers were first tested in a three-trial olfactory associative learning assay. For further tests, we selected only good learners that responded in two out of three conditioning trials. One group was tested in an additional olfactory associative learning assay involving different reward volumes and concentrations. Another group was tested for risk sensitivity in a two-alternative forced-choice PER procedure and then in a latent inhibition (LI) assay. Levels of acquisition in olfactory associative learning were highest in pollen and water foragers, and better acquisition was associated with collection of heavier pollen loads and smaller and lighter nectar loads of lower sugar concentration. Among the good learners, pollen foragers still showed better acquisition than nectar foragers when rewarded with several volumes and concentrations of sucrose solution. Pollen and nectar foragers were equally risk averse, preferring a constant reward to a variable one, and choice was not affected by pollen load weight. Communicated by M. Giurfa T. Drezner-Levy : S. Shafir (*) B. Triwaks Bee Research Center, Department of Entomology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel e-mail: [email protected] B. H. Smith School of Life Sciences, Arizona State University, Phoenix, AZ, USA

Contrary to a previous study, pollen and nectar foragers were similarly affected by LI. We discuss possible explanations for the discrepancy between the two studies. Overall, our results suggest that differences between foraging groups in sensitivity to various stimuli may not correspond to differences in choice behavior. Keywords Apis mellifera . Risk sensitivity . Latent inhibition . Proboscis extension conditioning

Introduction One of the remarkable aspects of a social insect colony is its ability to integrate work performed by individuals specializing in different tasks. A honey bee queen typically mates with upward of 10 drones so that there are several patrilines of workers in a colony (Winston 1987; Palmer and Oldroyd 2000; Tarpy et al. 2004). The probability of engaging in a particular task is genetically influenced by the worker’s patriline and is age dependent, with older individuals engaging in foraging activities. These include foraging for nectar, pollen, water, and propolis (Winston 1987; Seeley 1995). Foraging specialization also contains a genetic component, and colonies that hoard large amounts of pollen can be quickly selected for (Hellmich et al. 1985; Page and Fondrk 1995). Foraging specialization has been correlated with several behavioral traits (reviewed in Scheiner et al. 2004; Page et al. 2006). Bees in lines selected for high pollen hoarding are more responsive to sucrose than bees in lines selected for low pollen hoarding (Page et al. 1998; Pankiw and Page 1999; Scheiner et al. 2001a, b). Highly responsive individuals have low thresholds for responding to sucrose. When their antennae are stimulated with sucrose solution,

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highly responsive bees respond with proboscis extension response (PER) to lower sucrose concentrations than less responsive bees. The same pattern is observed in unselected colonies; pollen foragers are more responsive to sucrose than nectar foragers (Page et al. 1998; Pankiw and Page 2000; Scheiner et al. 2001b, 2003). Pollen foragers are not only more sensitive than nectar foragers to sucrose but also to other stimuli, including pollen, odors, and light (Page et al. 2006). Differences in sucrose sensitivity are correlated with differences in associative learning performance. When an olfactory or tactile conditioned stimulus is associated with an unconditioned sucrose reward over several conditioning trials, bees that are more sensitive to sucrose respond to the conditioned stimulus in a greater number of trials (Scheiner et al. 1999, 2001a, b, 2003; Scheiner 2004). Furthermore, bees with similar sucrose responsiveness show similar learning performance, regardless of genotype (Scheiner et al. 1999, 2001a, b, 2003). When bees with high sucrose responsiveness are rewarded with a low sucrose concentration, they show similar learning to bees with low sucrose responsiveness that are rewarded with higher sucrose concentration (Scheiner et al. 2004, 2005). It is well established that pollen foragers are more responsive than nectar foragers to sucrose and other stimuli and hence also perform better in various measures of associative learning. However, the performance of bees foraging exclusively for water or for both nectar and pollen has been studied very little. There appear to be similarities between bees foraging for pollen and those foraging for water and between those foraging for nectar and those foraging for both nectar and pollen. In lines selected for high pollen hoarding, a higher proportion of bees returning without pollen are water foragers than in lines selected for low pollen hoarding (Page et al. 1998). Pankiw and Page (2000) found that water foragers are even more responsive to sucrose than pollen foragers and that foragers for nectar or for both nectar and pollen are similarly unresponsive. However, that study included a relatively small sample size, especially of water foragers. One of the goals of the present study is to compare the associative learning performance of foragers for water, pollen, nectar, and both nectar and pollen. Conditioning of the PER has been a fruitful tool for studying associative learning in bees (Menzel and Bitterman 1983; Bitterman 1996) and specifically for comparing learning performance between nectar and pollen foragers (Scheiner et al. 2004; Page et al. 2006). In this procedure, the measured response is whether or not (yes–no) a subject extends its proboscis to a conditioned stimulus. It is sometimes possible to infer choice proportions between alternatives by the associative strengths of each of the alternatives measured independently by PER (Couvillon

Behav Ecol Sociobiol (2009) 64:135–148

and Bitterman 1985). However, this approach has its limitations (Williams 1994; Bitterman 2006). The effects of reinforcements can also be studied by choice measures. Experimental paradigms in which choice is measured directly are a useful complement for a better understanding of underlying cognitive processes (Williams 1994; Behmer et al. 2005). Furthermore, in stimulus discrimination assays, two-alternative forced-choice (2AFC) procedures are theoretically, and even more so empirically, more reliable than yes–no procedures (Macmillan and Creelman 1991; Marvit et al. 2003). Shafir et al. (1999) modified the PER conditioning paradigm to a 2AFC one, in which subjects choose between two conditioned stimuli. This combines the advantages of PER conditioning in terms of accurate control of experimental variables with those of a choice procedure. The 2AFC PER paradigm has been used to study risk sensitivity in honey bees (Shafir et al. 1999, 2005, 2008; Drezner-Levy and Shafir 2007). In a typical risk-sensitivity experiment, the subject has to choose between an alternative that provides a certain reward and an alternative that provides a variable reward. Risk sensitivity is a powerful tool for testing underlying processes of learning and choice behavior (Kacelnik and Bateson 1997; Shafir 2000; Shapiro 2000; Shapiro et al. 2001; Waddington 2001; Weber et al. 2004). Risk sensitivity is also a fundamental component of the decision-making process of foragers in the field since floral resources are generally variable (Shafir et al. 2003). Thus, another goal of the present study is to use the 2AFC PER paradigm to compare for the first time risk sensitivity of foragers of different tasks. This is part of a general recent effort to relate genetic traits that underlie learning and choice performance to decisions faced by foragers in the field (Latshaw and Smith 2005) and a contribution to characterizing behaviors associated with the pollen foraging syndrome (Page et al. 2006). Another learning paradigm of interest is latent inhibition (LI), in which subjects learn to ignore an unrewarded stimulus. Honey bees can be tested for LI using PER conditioning, and their performance in LI is correlated with other learning tasks and has been shown to contain a heritable component (Chandra et al. 2000, 2001; Ferguson et al. 2001). Recently, Latshaw and Smith (2005) found that pollen foragers learned better than nectar foragers to ignore an unrewarded odor in LI. This finding was in support of pollen foragers being generally more sensitive to stimuli since latent inhibition learning does not involve a sucrose reward. Therefore, differences in LI performance cannot be directly attributed to variation in sucrose responsiveness. Testing for LI requires to first exclude subjects that are not motivated to learn an odor-sucrose association. In order to maintain high variation in associative conditioning performance among subjects, Latshaw and Smith (2005)


set a low selection criterion and only excluded subjects that did not respond at all or responded only once in five conditioning trials. Since the number of responses during conditioning is strongly correlated with sucrose responsiveness (Scheiner et al. 1999, 2001a, b, 2003; Scheiner 2004), this selection criterion probably only excluded bees with very low sucrose responsiveness. Among the subjects tested for LI, there would be a large variation in sucrose responsiveness, and hence also in acquisition, with higher mean responsiveness and learning performance in the pollen than in the nectar foragers. Chandra et al. (2000, 2001) and Ferguson et al. (2001) used a stricter criterion and only selected subjects that responded in two of three conditioning trials. A third goal of this study was to compare LI of nectar and pollen foragers using this strict selection criterion. This would test for a correlated response between LI and foraging task in subjects that perform similarly in associative learning.

Methods

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foraging group according to the criteria of Page and Fondrk (1995). Bees that had pollen loads in their curbiculae and that regurgitated fewer than 5 μl were considered pollen foragers. Those without pollen loads and that regurgitated more than 5 μl fluid of at least 10 brix were considered nectar foragers. Bees that carried both pollen and 5 μl or more fluid of at least 10 brix were considered pollen and nectar foragers, and bees that carried 5 μl or more fluid of less than 10 brix were considered water foragers. Bees that returned empty, without nectar or pollen, were considered empty. We excluded this group from the main experiments. To avoid starvation, we fed bees 0.8 μl sucrose solution (35% w/w) 1 h after harnessing them. Ten percent of bees died before this feeding, and of those surviving, 6% did not feed and were excluded. After one more hour, we conducted a motivation test. We gently touched the antennae of each bee with a drop of sucrose solution (35% w/w) and only selected bees that extended their proboscides in response to the sugar stimulus; only 5% of bees did not pass this performance criterion. Testing with such a high sucrose concentration only excludes the most extremely unresponsive bees (Scheiner et al. 2005).

Restraint of subjects Apparatus Bees were maintained in standard honey bee hives at the apiary of the B. Triwaks Bee Research Center, Rehovot, Israel, and were free to fly and forage. Subjects in the various experiments came from four colonies of the New World Carniolan race (Cobey 1999). We harnessed subjects as in Shafir et al. (1999, 2005, 2008) and Drezner-Levy and Shafir (2007). Foragers were collected into small glass vials as they returned to the hive. To facilitate harnessing of the bees, each vial was submerged into ice water until the bee stopped moving (ca. 1 min). Subjects were then strapped into a sectioned hollow plastic tube by a 3-mm-wide strip of duct tape that wrapped around the tube and (dorsal) thorax of the bee. The abdomen of the bee was not covered. Subjects were harnessed so that the stand extended to just below the front pair of legs, which were loose over the stand, to ensure that the head of each bee was free to rotate. Harnessed bees were always kept in an air-conditioned room, with the thermostat set at 25°C, to ensure similar conditions during all of the experiments. Classification of bees to forager group About 30 min after harnessing, when the bees had recovered from the ice water, we gently squeezed the abdomen of every bee, collected the regurgitated contents of the crop with a micropipette, and measured its concentration with a refractometer. We scraped off pollen pellets of bees that had pollen in their curbiculae, and the pellets were dried and weighed (Sartorius CP225D model, ±0.01 mg). Bees were classified to

Odors (conditioned stimulus (CS)) were delivered to subjects from 1-ml glass syringes mounted at a training station. Overall, we used four odors that bees can learn equally well and discriminate well between them: eugenol, geranyl acetate, benzyl acetate, and 1-octanol (DreznerLevy and Shafir 2007). We added 3.5 μl of pure odor to a strip of filter paper that was placed inside a syringe. The tip of each syringe was attached by silicone tubing to a valve that was attached to an air pump. Valves were controlled by a computer, and opening of a valve caused an odor air stream flow of 13 cm3/s out of the tip of the syringe and over the subject’s antennae. To create an exhaust stream, we connected a 9-cm-diameter tube to a vent and mounted it 13 cm behind the subject. Thus, subjects experienced a constant slow air flow, and when a particular valve was opened, an air stream of the corresponding odor flowed over the antennae of a subject and immediately into the exhaust stream. Subjects were lined up on a ruler, at 4-cm intervals, with a partition between each subject and its neighbors. After a trial with one subject, the ruler was slid until the next bee was in position, and so forth. Procedure The sequence of experimental assays performed is shown in Table 1. Since one of our goals was to test bees in a LI assay, the first conditioning procedure for all bees involved

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Table 1 Sequence of experimental assays performed Day 1

Classification to forager group

Day 2: latent inhibition

Selection in 3-trial conditioning assay

Effect of reward volume and concentration Risk sensitivity Risk-sensitivity sham

Phase I

Phase II

30 unrewarded trials 30 unrewarded trials No odor exposure control

6 rewarded trials 6 rewarded trials 6 rewarded trials

On day 1, all bees were classified to forager group and tested in a three-trial conditioning assay. Subjects that passed the selection criterion were divided into three groups. One group was tested with various reward volumes and concentrations. A second group was tested in a risk-sensitivity assay and the following day in a latent inhibition assay, consisting of a first phase of 30 unrewarded trials, followed by a second (test) phase of six rewarded trials. A third group went through a sham risk-sensitivity assay on the first day and then divided into two subgroups the following day. One subgroup was tested in a latent inhibition assay, and the second subgroup acted as control and was not exposed to odor during phase I and was then tested in six rewarded trials in phase II

forward pairing of an odor with sucrose reinforcement (Chandra et al. 2000; Latshaw and Smith 2005). This phase selects subjects that are motivated to respond to the US and learn the association of odor with it. We used eugenol odor for the conditioned stimulus. There were three conditioning trials, with an intertrial interval (ITI) of 8 min. A trial began when a bee was placed in the training station. We allowed the subject a few seconds to acclimate, and then we presented the odor for 5 s. After 3 s, we presented the unconditioned stimulus, 0.4 μl of a 35% w/w sucrose solution. We noted whether the subject extended its proboscis after the onset of odor delivery, but before delivery of the reward. We lightly touched the subject’s antennae with the tip of the syringe to induce PER, and the subject was allowed to imbibe the sucrose reward; subjects always ingested the entire droplet. Once a subject learned the association and extended its proboscis after odor presentation, we brought the tip of the syringe directly to the tip of the proboscis. We used the selection criterion of Chandra et al. (2000), selecting for further experiments only bees that responded in two of the three trials. After the three-trial conditioning assay, subjects were divided into three groups. One group was tested for the effect of reward volume and concentration on learning performance. A second group was tested in a risk-sensitivity assay later on the first day and then for LI on the following day. A third group went through a sham risk-sensitivity assay on the first day and then split into two subgroups the following day, one tested for LI and one acting as a control for LI. Group 1: effect of reward volume and concentration The goal of this assay was to compare the learning performance of foraging groups when reinforced with a range of sucrose solution volumes and concentrations. This procedure included many experimental conditions, and the proportion of foragers collecting water or both nectar and

pollen was relatively small. We therefore concentrated only on foragers collecting nectar or pollen. For the conditioned stimulus, we used geranyl acetate for half of the subjects and benzyl acetate for the other half, balanced between experimental conditions. We tested four reward volumes of a 35% w/w sucrose solution (0 (no reward), 0.1, 1, or 4 μl) and four reward concentrations of a 0.8 μl sucrose solution (0% (distilled water), 5%, 25%, or 55%). The procedure was the same as above, except that there were six conditioning trials. Group 2: risk sensitivity The risk-sensitivity phase consisted of choice trials between two odors, using the 2AFC PER paradigm (Shafir et al. 1999, 2005, 2008; Drezner-Levy and Shafir 2007). The two odors were benzyl acetate and geranyl acetate. We attached each of two odor syringes to a base that mounted on to tracks at the training station. Syringes were mounted horizontally so that when we placed a subject in the training station, the tips of the syringes were 10 mm from the bee and pointed toward the base of the bee’s antennae. Each subject was positioned so that syringes were 30° to the right and the left of its sagittal plane. A line drawn on the base of the station defined the midline between the syringes. For each subject, one odor was associated with a constant reward volume and the other with a variable reward volume. Reward concentration was 35% w/w sucrose solution. The odor assigned to the constant reward was counterbalanced among subjects to control for possible preferences for odors that were not due to associated rewards. The two odors were presented in an alternating, pulsed schedule. The schedule consisted of 0.8 s of one odor, followed by 0.2 s of no odor, followed by 0.8 s of the other odor, and so forth, until each odor was presented twice. We alternated the order in which the two odors were pulsed across trials to


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control for a possible sequence effect of odor presentation. To control for possible side preferences, we presented each odor on the left (L) or right (R) in the sequence RLLRLRRL...LRRL. The other odor was always presented from the opposite side. We scored the orientation of the head of each subject with respect to the midline between the two syringes after the last of the four odor pulses, when the computer emitted an audible signal. We scored a choice on every trial, even if the head of a bee showed only a slight deviation from the midline. A video camera mounted above the training station facilitated scoring of choices. The chosen odor was presented for an additional 3.5 s, and the subject was rewarded 1.5 s after the onset of odor. We delivered rewards regardless of whether or not a subject extended its proboscis to the chosen odor. Each subject was tested in one of two experimental conditions (Table 2). For a zero reward, we gently touched the subject’s antennae with the tip of a clean syringe and allowed it to touch the empty syringe if it extended its proboscis. The sequence of low and high rewards in the variable alternative was predetermined for each condition according to the appropriate probabilities and distributed across the 24 trials in a regular manner. For half of the subjects, the sequence started with high reward, followed by low reward(s), then high again, and so forth, and for the other half of the subjects, the sequence started with low reward(s), followed by high reward, then low reward(s) again, and so forth. Every time a subject chose the odor that corresponded to the constant reward, it received the appropriate amount of sucrose solution. The first time that a subject chose the variable reward, it received the first value in the sequence. The next time that the subject chose the variable reward, it received the next value in the sequence, and so forth. Thus, every subject within an experimental condition experienced a similar probability as other subjects of low and high values of the variable reward. We conducted 24 trials with each subject, with an ITI of 6 min. We then calculated the amount of sucrose solution that each bee imbibed during the experiment, which depended on the choices she made, and on the experimental

Table 2 The values of the constant and variable rewards volumes (μl) in each condition of the risk-sensitivity assay and the values of the corresponding mean, variance, and coefficient of variation (CV) Condition

1 2

Constant reward

0.5 1

Variable reward Low (prob.)

High (prob.)

0 (0.8) 0 (0.5)

2.5 (0.2) 2 (0.5)

Mean

Variance

CV

0.5 1

1 1

200 100

The probabilities of low and high variable rewards are in parentheses

condition. We fed each bee the necessary volume of a 35% w/w sucrose solution to reach a total feeding of 25 μl during the entire experiment. This was designed to equalize the energetic state of all subjects toward the LI assay on the following day and to allow them to survive the night. The bees were kept in a dark room for 17.5 h, until the next morning. Those that survived (88% of all bees) were then tested for LI. Group 2: latent inhibition Phase I of the LI assay consisted of exposure to an unrewarded odor for 30 trials. Up to 12 bees were lined up on a ruler, at 4-cm intervals, with a partition between each subject and its neighbors. In front of each bee was a 1-ml glass syringe loaded with 3.5 μl of 1-octanol pure odor. The tip of each syringe was attached by silicone tubing to a valve that was attached to an air pump and controlled by a programmable controller (Direct logic DL05). To create an exhaust stream, we connected a 9-cm-diameter tube to a vent and mounted it 36 cm behind the subjects. A funnel-shaped Plexiglass box shunted the air from the odor syringes toward the exhaust tube. Each odor presentation lasted 4 s, with an ITI of 6 min. Phase I was conducted in a different room than where the other assays were conducted to reduce nonassociative effects, such as generalization to the conditioning station. Phase I lasted 3 h, and phase II began 10 min later. Phase II tested whether an inhibition to the conditioned odor was learnt during phase I. It involved forward pairing of 1-octanol with a 0.4 μl sucrose solution of 35% w/w. We presented the odor for 7 s, and after 4 s, we presented the sucrose solution to the bee. There were six trials, with an ITI of 5 min. Group 3: risk-sensitivity sham and latent inhibition control We wanted to test whether the responses of subjects during phase II of the LI assay were in fact affected by the nonrewarded exposure to the odor during phase I and whether LI learning occurs with subjects that had been harnessed for 2 days and been tested in the risk-sensitivity assay on the previous day. We therefore first subjected bees to a sham risk-sensitivity assay on the first day, similar to the risk-sensitivity assay described above. There were 24 trials with an ITI of 6 min. In each trial, we presented either benzyl acetate or geranyl acetate, alternating between the two odors. We presented the odor for 5 s, and after 3 s, we presented the bee 0.5 μl of a 35% w/w sucrose solution. The next day, we randomly split the bees into two subgroups. One subgroup was exposed to the odor during phase I as above, and the other half waited the same amount of time but with no odor exposure. Both subgroups were then tested in phase II as above.

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We used the likelihood ratio Chi-square test to compare responses between foraging groups during the pre-selection phase. We also used this test to compare spontaneous responses on the first trial in associative conditioning assays. We used the Wilcoxon rank-sum test (equivalent to Mann–Whitney U test) for one-way analysis of the number of responses on later trials. For two-way analysis of the proportion of spontaneous responses on the first trial, we used a nominal logistic regression. When the dependent variable was a discrete response, such as the total number of trials in which a subject responded with PER to the CS, the data did not conform to requirements of a parametric ANOVA. We ranked the dependent variable and compared the performance of different foraging groups using one- or two-way nonparametric ANOVA on ranks (Conover and Iman 1981). Pollen and nectar load measurements were continuous and were analyzed by regular ANOVA. We performed multiple comparison post tests using Tukey’s method. We also report Pearson product-moment correlation coefficients between pollen and nectar load measurements and number of responses in a learning assay. All analyses were performed with JMP 7 (SAS Institute) software.

Results Pre-selection assays We assayed returning bees for this and another study (Drezner-Levy and Shafir 2007) concurrently. Overall, 9.9% of bees (n=6,179) died before the first feeding about 1 h after collection, and there were differences between foraging groups (Chi-square test: X2 =94.2, df=4, p