The Effect of Insecticides Considered Harmless to Honey Bees (Apis ...

BEHAVIOR

The Effect of Insecticides Considered Harmless to Honey Bees (Apis mellifera): Proboscis Conditioning Studies by Using the Insect Growth Regulators Tebufenozide and Diflubenzuron C. I. ABRAMSON,1 JON SQUIRE,1 AUDREY SHERIDAN,2

AND

PHILLIP G. MULDER, JR.2

Environ. Entomol. 33(2): 378Ð388 (2004)

ABSTRACT Experiments were designed to examine the effects of the IGRs tebufenozide and dißubenzuron on Pavlovian conditioning of harnessed foragers. In one set of experiments, animals learned a simple task in which they associated a conditioned stimulus with feeding. A second set of experiments required the animal to learn a complex discrimination task. Within each experiment, separate groups of bees were pretreated with the insecticide, whereas others received the insecticide imbedded in the sucrose solution used as the unconditioned stimulus. Results indicated that exposure to both tebufenozide and dißubenzuron inßuence the performance of honey bees. Bees pretreated with 10 ␮l of tebufenozide, 10 min before learning a simple Pavlovian task, produced lower levels of acquisition when the concentrations were administered in dosages equivalent to 0.22, 0.29, and 1.0 liter/ha. When animals were given a series of 1-␮l droplets of dißubenzuron over the course of 12 training trials, the effect on acquisition and extinction was even more pronounced. No such effects were seen when tebufenozide was used. Discrimination learning was also inßuenced in bees pretreated with the insecticides or having the insecticides imbedded in the unconditioned stimulus. KEY WORDS honey bee, learning, insecticides, Pavlovian conditioning, proboscis extension

THE USE OF PAVLOVIAN CONDITIONING as a sensitive and reliable bioassay to test the effect of lethal and sublethal levels of agrochemicals on behavior is well established (Taylor et al. 1987, Mamood and Waller 1990, Decourtye and Pham-Dele` gue 2002, Weick and Thorn 2002). Studying the inßuence of sublethal amounts of insecticides on honey bee, Apis mellifera L., behavior is important for the survival of honey bees, public policy issues, honey bee population regulation, environmental degradation, and the use of biological controls. What is seldom explored in the literature are experiments speciÞcally designed to survey the effects of insecticides speciÞcally known not to “harm” honey bees. These compounds may include some of the new generation pyrethroids, insect growth regulators (IGRs), and fermentation by-products, all of which are currently used in formulation of new products. Many of these new products are considered by the Environmental Protection Agency and other regulatory bodies as user-friendly, target-speciÞc, and environmentally safe. However, little is known about their effects, if any, on honey bee behavior, particularly learned behavior. To use these chemicals effectively and without injuring these important pollinators, it is 1 Laboratory of Comparative Psychology and Behavioral Biology, Departments of Psychology and Zoology, Oklahoma State University, Stillwater, OK 74078. 2 Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078.

important to know what effects these agrochemicals have on honey bee behavior. In addition to providing data on the effect of “harmless” chemicals on honey bee behavior, our work takes on added signiÞcance when we consider that it is possible to seek “fast-track” approval for exemption labeling. Many of the compounds we are studying fall under this category. We consider this a potentially dangerous precedent because the effects of these fasttrack materials on the complete spectrum of honey bee behavior are unknown at this time. Our Þrst experiments on the study of chemicals considered not harmful to honey bees was an investigation of dicofol. This product is a chlorinated hydrocarbon insecticide and a chemical analog of DDT. It is considered nontoxic to most insects and is used primarily as an acaricide. We used the proboscis conditioning paradigm to detect any deleterious effects of the insecticide on learning. Training consisted of pairing a neutral olfactory cue (conditioned stimulus) with sucrose (unconditioned stimulus) to produce a learned response to the conditioned stimulus alone. Each bee received 12 acquisition trials followed by 12 extinction trials in which the unconditioned stimulus was omitted. To control for pseudoconditioning, unpaired animals received an equal number of conditioned stimulus/unconditioned stimulus presentations in a pseudorandom sequence. The toxic effects from insecticides were observed in the inability of bees to learn to extend their proboscis to an olfactory

0046-225X/04/0378Ð0388$04.00/0 䉷 2004 Entomological Society of America

April 2004

ABRAMSON ET AL.: INSECTICIDES CONSIDERED HARMLESS TO HONEY BEES

cue. Much to our surprise, we discovered that honey bees pretreated with dicofol exhibited signiÞcantly lower levels of learning than honey bees not pretreated (Stone et al. 1997). The purpose of the present experiment is to examine the effects of the IGRs tebufenozide and dißubenzuron on Pavlovian conditioning of harnessed foragers. These insecticides were chosen on the basis of their extensive usage on honey bee-foraged crops such as cotton and alfalfa. Tebufenozide is a molting disruptor, and its mode of action is to mimic the natural insect molting hormone 20-hydroxyecdysone. When ingested by targeted insect larvae, the insecticide acts as an agonist in the chemical processes concerned with cuticle formation. A new but malformed cuticle is created leading to an excess of chitin under the exoskeleton and eventual death by dehydration and starvation (Walgenbach 1999). The active ingredient is tebufenozide, which is described as “being safe for pollinators such as the honey bee” (Dhadialla et al. 1998). This statement referred to a Þeld study that recorded the number of foraging bees, dead adults, and dead pupae after exposure to 10 times the recommended dose. The results indicated that exposure did not kill or disrupt foraging behavior and had no effect on either eclosion or larval development of honey bees (Heller et al. 1992). Dißubenzuron is a chitin inhibitor and disrupts the synthesis of the chitin exoskeleton that is required before a larval molt. The new cuticle is weakened and leads to an unsuccessful molt and eventual death. There is also a secondary effect in which eclosion is prevented, or if larvae emerge, die shortly thereafter (Tripathi 1996). The active ingredient is described as having no effect on honey bees after exposure (Tripathi 1996). This report fails to state how the toxicity of the insecticide was measured. A more detailed investigation reviewed studies that used rates ranging from 30 ␮g per bee for oral and contact LD50 values (IPSC Health and Safety Guide No. 99, 1995) to 115 ␮g per bee LD50 values (Information Ventures, Inc. 1995). The IPSC study noted that colonies were not affected after aerial application of 350 g dißubenzuron/ha. The amount of insecticide delivered to bees was determined by labeled rates for a ground application. Six rates were chosen representing the highest, lowest, and three intermediate concentrations that may be applied in an intensive pest control program. These labeled rates were then diluted with equal parts of a 1.8 M sucrose solution to improve palatability and to compensate for chemical repellency (Thompson 2003). Therefore, the quantities of insecticide fed to bees are representative of a 50% dilution of the labeled rates. Materials and Methods Overview of Experimental Design. Two types of conditioning experiments were performed. In both experiments, one of six levels of an insecticide was applied 10 min before training or during training.

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When an insecticide was administered during training it was imbedded in the unconditioned stimulus (US). The rationale behind the application of the insecticide was to explore the effect of a single dose (administered before training) or of several smaller doses (administered during training in US presentations) on learning in honey bees. Bees were fed the insecticides via a Hamilton microsyringe. As a control against experimenter bias, the insecticides were placed in containers identiÞed only by a number 1 through 6. A conditioning trial began by picking up a bee and placing it in front of a ventilation fan. The purpose of the fan was to remove conditioned stimulus (CS) and US scents from the training area. Several seconds after being placed in front of the fan, the appropriate stimulus was introduced. After application of a stimulus, the animal was returned to a holding area and a second animal was run. This continued until all the animals scheduled to be run on that day received the required number of training trials. An attempt was made to control for calendar variables and ßuctuating hive conditions by running animals from several groups daily. Experiment 1 investigated the effects of tebufenozide and dißubenzuron on simple Pavlovian conditioning where honey bees were trained to associate a CS with a US. After a 12-trial acquisition phase, an extinction phase was initiated in which the CS was presented 12 times without the US. The rationale for including an extinction phase was to determine whether the insecticide inßuenced persistence of a conditioned response when the response was no longer followed by a feeding. The performance of paired animals was evaluated with an “unpaired” group of animals receiving an identical number of CS and US presentations that were explicitly unpaired. The unpaired group was included to ensure that any learning observed in the insecticide groups was actually the result of the pairing between the CS and US and not due to factors such as central excitatory state in which the honey bee extends its proboscis to the presentation of an olfactory stimulus because it has been sensitized by a sucrose feeding (Abramson 1994). In addition to serving as a control for the paired groups, we also wanted to know whether the insecticides inßuenced unpaired responses. This would be revealed by a high level of CS responding in unpaired animals treated with the same insecticide concentrations as their match paired group. Experiment 2 investigated the effects of tebufenozide and dißubenzuronon on complex Pavlovian conditioning where honey bees were trained to discriminate between two CSs, one of which was always paired with a US. The rationale behind including a discrimination experiment was to determine whether exposure to the insecticide inßuences different types of learning in the honey bee. We consider discrimination learning to be more complex than the single CS experiment because the honey bee must discriminate between two stimuli. The discrimination experiment also served as a check for the paired/unpaired experiments because in discrimination, each honey bee acts

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as its own control. It should be noted that the honey bee is also required to discriminate in the simple learning situation. The discrimination in the simple conditioning experiment is between the explicit CS used in training and all background stimulation. Preparation of CS and US Delivery Devices. In the simple learning situation, the CS consisted of exposure to cinnamon odor (GilbertieÕs, Southampton, NY). In the complex learning situation, one CS consisted of cinnamon odor and the second CS consisted of the odor of citral (product number C-1645, Sigma, St. Louis, MO). The odors of cinnamon and citral were selected for the discrimination experiment because previous work indicated that bees consider these stimuli equally salient (Abramson et al. 1996). The CS scent was Þrst transferred to a 1-cm2 piece of Þlter paper (Whatman no. 4) by using a wooden dowel. The paper was dabbed with scent to the point of saturation and then secured to the plunger of a 20-ml plastic syringe with a metal thumbtack. The US was either a 1.8 M sucrose feeding or one of six levels of insecticide. One of these levels was a sucrose only control. To induce the bees to ingest the insecticide, it was diluted in 1.8 M sucrose and fed directly to the honey bee with a Hamilton microsyringe. The rationale behind this method was to ensure that all bees were exposed to identical amounts of the insecticide. We also wanted to mimic a situation in which a honey bee consumed insecticide-laced nectar. The microsyringe was used whenever animals were pretreated with insecticide solution (or sucrose) and when an insecticide was embedded in a sucrose US. In experiments where the insecticide solution was administered before training, there was no need for the microsyringe, and the US was administered on Þlter paper strips dipped in 1.8 M sucrose. The Þlter paper strips were handled with tweezers. Capturing and Harnessing Honey Bees. Worker honey bees were selected randomly from the exterior surface and landing board of a single laboratory colony (Stillwater, OK) between May and November 2001. Bees collected in this way are a mixture of different behavioral specializations requiring either departure from the hive (i.e., foragers or nest cleaning bees) or remaining near the entrance (i.e., guards). They were captured individually in glass vials ⬇7:00 a.m. on the day before use and taken to the laboratory, where the vials were placed in an ice water bath. No attempt was made to determine the age of the subjects. When the bees became inactive to permit handling, they were removed from the vials and secured in individual restraining harnesses constructed from 0.38 caliber shells with a small strip of duct tape placed between the head and thorax and fastened to the sides of the harness. Once harnessed, subjects were fed a 1.8 M sucrose solution until satiated and left overnight to ensure that all subjects would have approximately the same motivation to feed during training the following morning. Experiment 1: Simple Pavlovian Conditioning. One thousand two hundred honey bees were divided into two equal-sized groups: 600 bees were used in the

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tebufenozide experiments, and the remaining 600 were used in the dißubenzuron experiments. The two 600-bee subgroups were further divided into two groups of 300 bees each. One of these groups received paired CS-US presentations, and the other group received unpaired CS or US presentations and served as controls for pseudoconditioning. Both groups of 300 bees were further divided into two groups of 150 bees each. For one group of 150, bees received 10 ␮l of insecticide solution 10 min before training. The remaining 150 bees received 10 ␮l of sucrose solution 10 min before training and received 1 ␮l of the insecticide solution imbedded in the US. Each group of 150 bees was further divided into six groups of 25 bees each. These six groups corresponded to the six levels of insecticide that are based on a 186.94-liter/ha spray volume, as administered by a ground applicator. Those in the tebufenozide groups received rates of 0, 0.15, 0.22, 0.29, 0.55, or 1.0 liter/ha, corresponding to active ingredient levels of 0, 16, 24, 32, 69.4, and 131 ␮g per bee. Bees in the dißubenzuron groups received 0, 0.027, 0.07, 0.15, 0.29, or 0.55 liter/ha, corresponding to active ingredient levels of 0, 3.4, 8.5, 16, 32, and 69.4 ␮g per bee. Animals in the paired groups received 12 acquisition trials followed by 12 extinction trials. A nonoverlap procedure was used in which the CS terminated before the US was presented. The CS was a 3-s presentation of cinnamon odor; the US duration was a 2-s feeding when Þlter paper strips were used or alternatively, the consumption of the entire 1-␮l droplet when the micosyringe was used. Animals typically consumed the droplet in ⬇2 s. The interstimulus interval (time between the onset of the CS and onset of the US) was 3 s. When a nonoverlap procedure is used, the interstimulus interval is identical to the CS duration. The intertrial interval (ITI, the time interval between the end of the US and the beginning of the CS) was 10 min. Animals in the unpaired groups received 12 CS presentations and 12 US presentations in a pseudorandom order. For one-half the unpaired animals, stimulus presentations consisted of three successive sequences of CS US US CS US CS CS US. For the remaining animals, the sequence consisted of US CS CS US CS US US CS. The interval between stimulus presentations was 5 min, or one-half the time than that used for the paired animals. The rationale behind using a 5-min ITI for unpaired animals was to keep the time between CS presentations ⬇10 min. If a 10-min ITI was used, the time between CS presentations would be ⬇20 min, and any difference between paired and unpaired animals might be accounted for in terms of such nonassociative effects as the time spent harnessed. After the 12 CS and US presentations, the unpaired experiment was terminated (no extinction trials). Responses to the CS were visually categorized into one of two states during each trial. If a subject extended its proboscis after the onset of the CS, but before the US was presented, a response was registered. Otherwise, a nonresponse was recorded.

April 2004 Table 1.


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Statistical summary for tebufenozide and diflubenzuron in simple learning experiments Pretreated

Treated in US

Amt. tebufenozide, ␮g/bee

Paired/unpaired vs. trial

Paired/unpaired


Paired/unpaired

0 15.975 23.965 31.96 69.35 130.7

10.796* 14.971* 3.6* 3.638* 7.751* 3.517*

265.168* 226.923* 72.849* 20.344* 260.612* 59.272*

17.73* 5.324* 4.109* 7.112* 4.227* 5.816*

162.609* 64.297* 27.066* 48.604* 38.636* 57.604*

Amt. dißubenzuron, ␮g/bee


Paired/unpaired


Paired/unpaired

0 3.41 8.53 15.975 31.96 69.35

10.024* 2.638* 5.9216* 7.517* 2.995* 1.043

90.231* 25.693* 27.898* 56.898* 34.605* 10.235

4.235* 0.717 1.925 1.422 2.495* 1.63

38.923* 13.82* 18.343* 2.489 8.319* 9.184*

Pretreated

Treated in the US

Note: due to the nature of the experiment, the paired/unpaired responses relate only to the acquisition phase. Degrees of freedom for paired/unpaired vs. trial were 11, 528. Degrees of freedom for paired/unpaired were 1, 48. * P ⬍ 0.05.

Experiment 2: Complex Pavlovian Conditioning. The design differed from that used in the simple learning experiment in that two CSs were used, one of which was paired with a feeding, and extinction was not tested. In addition, there was no unpaired group. The unpaired group is unnecessary in discrimination experiments because each subject serves as its own control. One CS was the odor of cinnamon used in the simple learning experiments, and the second was the odor of citral. The CS followed by a feeding was the CS⫹ and the CS not followed by a feeding was the CS⫺. For one-half the animals, CS⫹ was cinnamon and the CS⫺ citral; for the remaining animals, the CS⫹ was citral and the CS⫺ cinnamon. Six hundred and twenty-four honey bees were divided into two main groups. Three hundred twelve bees were used in the tebufenozide experiments and the remainder was used in the dißubenzuron experiments. Both groups of 312 bees were further divided into two groups of 156 bees each. For one group of 156, bees received 10 ␮l of insecticide solution 10 min before training. The remaining 156 bees received 10 ␮l of sucrose 10 min before training and received 1 ␮l of the insecticide solution imbedded in the US. Each group of 156 bees was further divided into six groups of 26 bees each. These six groups corresponded to the six levels of insecticide used in experiment 1. Acquisition consisted of 12 presentations of both the CS⫹ and CS⫺ so that 24 trials in total were conducted. For 13 of the 26 animals, the presentation of the CSs consisted of three successive sequences of CS⫹ CS⫺ CS⫺ CS⫹ CS⫺ CS⫹ CS⫹ CS⫺. For the remaining animals, CS⫹ and CS⫺ presentation was reversed. Learning was considered to have taken place if the bee extended its proboscis after the onset of the CS⫹ but before presentation of the US, and if the bee stopped responding to the CS⫺, which was not paired with a feeding. As in the previous experiment, a nonoverlap procedure was used. The CS duration was 3 s and the US

duration 2 s. The intertrial interval was reduced from 10 min to 5 min. The rationale behind using a 5-min ITI in the discrimination experiments was to keep the time between CS⫹ presentations ⬇10 min. If a 10-min ITI was used, the time between CS⫹ presentations would be ⬇20 min, and any difference between our simple and complex learning experiments can be accounted for in terms of such nonassociative effects as time spent harnessed. Responses to the CS were visually categorized into one of two states during each trial. If a subject extended its proboscis after the onset of the CS, but before the US was presented, a response was registered. Otherwise, a nonresponse was recorded.

Results Statistical analyses for all experiments were performed with SPSS using the General Linear Model with repeated measures. The F values for the analysis of variance (ANOVA) tests are presented in the text and in Tables 1 and 2. Before analysis, the raw data were transformed to the proportion of animals responding on each trial. Simple Pavlovian Learning. Tebufenozide. Fig. 1 shows the acquisition and extinction results in animals pretreated with 10 ␮l of tebufenozide solution across the six concentrations. The performance of animals receiving 1 ␮l of tebufenozide solution during each of the 12 acquisition trials across the six concentrations is in Fig. 2. The unpaired curves are not shown but they are consistent with published research (Abramson et al. 1997). Statistically signiÞcant (P ⬍ 0.05) differences were obtained between each paired group and its matched unpaired group for all of the insecticide concentrations, whether the animals were pretreated with tebufenozide or received it in the US (Table 1). This pattern of results indicates that animals in the paired groups learn to associate a CS with a sucrose feeding and that unpaired animals do not

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Table 2.

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Statistical summary for tebufenozide and diflubenzuron in complex learning experiments Pretreated

Treated in US

Amt. tebufenozide, ␮g/bee

CS⫹ vs. CS⫺ ⫻ Trial

CS⫹ vs. CS⫺

CS⫹ vs. CS⫺ ⫻ trial

CS⫹ vs. CS⫺

0 15.975 23.965 31.96 69.35 130.7

6.579* 5.01* 9.509* 2.431* 2.691* 2.38*

136.662* 48.226* 74.203* 15.248* 63.947* 19.645*

10.704* 3.11* 4.761* 4.046* 5.242* 3.318*

107.556* 44.198* 51.103* 27.977* 46.356* 24.568*

Amt. dißubenzuron, ␮g/bee


CS⫹ vs. CS⫺


CS⫹ vs. CS⫺

0 3.41 8.53 15.975 31.96 69.35

3.902* 11.025* 6.595* 3.650* 4.46* 2.76*

35.862* 50.884* 58.383* 33.547* 46.249* 25.935*

3.399* 1.663 1.771 1.168 1.343 1.701

31.449* 21.115* 13.982* 33.193* 7.058* 9.578*

Pretreated

Treated in US

Degrees of freedom for all tests were 1, 275. * P ⬍ 0.05.

become sensitized after consumption of various tebufenozide concentrations. It is interesting to note that Fig. 1 shows the acquisition results are split into two deÞnite groups. The Þrst grouping contains animals given 0, 0.15, or 0.55 liter/ha tebufenozide. The second grouping contains animals given 0.22, 0.29, or 1.0 liter/ha tebufenozide. The difference between the two groupings is signiÞcant (F ⫽ 8.544; df ⫽ 1, 144; P ⬍ 0.05), and there is a signiÞcant trial ⫻ concentration interaction (F ⫽ 1.765; df ⫽ 11, 1584; P ⬍ 0.05). In contrast to the performance of animals pretreated with a single10-␮l drop of tebufenozide, honey bees receiving 12 1-␮l drops over the course of training show no signiÞcant differences in acquisition. There was no signiÞcant effect of concentration (F ⫽ 2.211;

df ⫽ 1, 144; P ⬎ 0.05), nor was there a signiÞcant trial ⫻ concentration interaction (F ⫽ 0.907; df ⫽ 11, 1584; P ⬎ 0.05). The extinction results reßect those seen in acquisition. The second panel of Fig. 1 shows that animals pretreated with 10 ␮l of tebufenozide gradually reduce the number of CS responses when such responses are no longer followed by a US. This decline, however, is not statistically signiÞcant (F ⫽ 1.087; df ⫽ 11, 1,584; P ⬎ 0.05), but the number of CS responses elicited during extinction across the six concentrations is signiÞcant (F ⫽ 4.7; df ⫽ 1, 144; P ⬍ 0.05). A similar decline is found in the second panel of Fig. 2, where animals receive a series of 1-␮l doses of tebufenozide during acquisition but not in extinction. The decline was not statistically signiÞcant (F ⫽ 1.121; df ⫽ 11,

Fig. 1. Mean proportion of bees responding to the CS over the course of 12 acquisition and 12 extinction trials after a 10-␮l feeding of tebufenozide. The change from acquisition to extinction occurred on trial 13.

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Fig. 2. Mean proportion of bees responding to the CS over the course of 12 acquisition and 12 extinction trials. The US contained 1 ␮l of tebufenozide. The change from acquisition to extinction occurred on trial 13.

1,584; P ⬎ 0.05), nor did the number of CS responses signiÞcantly differ across the concentrations (F ⫽ 1.130; df ⫽ 1, 144; P ⬎ 0.05). We believe the decline in responses to the CS in both experiments would have reached signiÞcance given more extinction trials. Diflubenzuron. Fig. 3 shows the acquisition and extinction results in animals pretreated with 10 ␮l of dißubenzuron across the six concentrations. The performance of animals receiving 1 ␮l of dißubenzuron during each of the 12 acquisition trials across the six concentrations is presented in Fig. 4. The unpaired curves are not shown, but they are consistent with published research (Abramson et al. 1997). Statisti-

cally signiÞcant differences were obtained between each paired group and its matched unpaired group for all of the insecticide concentrations, whether the animals were pretreated with dißubenzuron or received it in the US (Table 1). Such a pattern of results indicates that animals in the paired groups learn to associate a CS with a sucrose feeding and that unpaired animals do not become sensitized after consumption of various concentrations of dißubenzuron. A comparison of Figs. 3 and 4 with the tebufenozide results reveals some intriguing differences between dißubenzuron and tebufenozide. The lower level of acquisition shown in animals given a single 10-␮l drop-

Fig. 3. Mean proportion of bees responding to the CS over the course of 12 acquisition and 12 extinction trials after a 10-␮l feeding of dißubenzuron. The change from acquisition to extinction occurred on trial 13.

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Fig. 4. Mean proportion of bees responding to the CS over the course of 12 acquisition and 12 extinction trials. The US contained 1 ␮l of dißubenzuron. The change from acquisition to extinction occurred on trial 13.

let of tebufenozide (Fig. 1) was also found with dißubenzuron (Fig. 3), although the results are less dramatic in animals pretreated with dißubenzuron. There was a signiÞcant effect of concentration (F ⫽ 2.704; df ⫽ 1, 144; P ⬍ 0.05) and no trial ⫻ concentration interaction (F ⫽ 1.286; df ⫽ 11, 1,584; P ⬎ 0.05). Acquisition was poorer in animals pretreated with a 0.55 liter/ha concentration, where approximately onehalf the bees responded to the CS. In extinction, the group exposed to a 0.55 liter/ha concentration was the only one showing a signiÞcant decline in CS responses when the US was discontinued, although, as in the tebufenozide experiments, there was a decline (nonsigniÞcant) in CS responses in all groups. Figure 4 clearly shows that acquisition is affected in animals given a series of 1-␮l drops over the course of 12 acquisition trials. All groups respond to the CS within the Þrst three trials. However, with the exception of the paired control group (no insecticide consumed), the conditioned responses begin to decline. The decline does not reach the level of the matched unpaired control groups, suggesting that animals consuming dißubenzuron are still able to learn but not at the level of honey bees that are insecticide free. The interaction between concentration and training trials is signiÞcant (F ⫽ 1.9; df ⫽ 11, 1,584; P ⬍ 0.05), and there are no signiÞcant differences between the concentrations (F ⫽ 2.094; df ⫽ 1, 144; P ⬎ 0.05). It should also be noted that the acquisition curves in the control group are not as high as in previous studies. Why the lower level of performance in control animals compared with our previous experiments is unknown at this time and may reßect some underlying hive condition and/or the operation of calendar variables. The extinction results are also interesting. The second panel of Fig. 3 shows that animals pretreated with 10 ␮l of dißubenzuron reduce the number of condi-

tioned responses when they no longer are paired with a US. This trend, as in the case with animals pretreated with 10 ␮l of tebufenozide, is not signiÞcant (F ⫽ 1.237; df ⫽ 11, 1,584; P ⬎ 0.05). However, the extinction performance across the six concentrations was different among the groups prefed 0, 0.027, 0.07, 0.15, 0.29, and 0.55 (F ⫽ 2.461; df ⫽ 1, 144; P ⬍ 0.05). The greatest decline during extinction occurred in the group given 0.55 liter/ha. The most signiÞcant extinction results are shown in the second panel of Fig. 4 where animals receive the insecticide in the US. The initial period of acquisition is followed by a decline in responding. This low level of response continued during the extinction phase. There was no signiÞcant concentration ⫻ trial interaction (F ⫽ 0.987; df ⫽ 11, 1,584; P ⬎ 0.05), nor was there a signiÞcant difference among the concentrations (F ⫽ 2.114; df ⫽ 1, 144; P ⬎ 0.05). The extinction results for animals given dißubenzuron in the US are very different from those observed in the tebufenozide experiments and in animals prefed dißubenzuron. Complex Pavlovian Learning. Tebufenozide. Fig. 5 shows CS⫹ responding in honey bees pretreated with 10 ␮l of tebufenozide across the six concentrations. The performance of animals receiving 1 ␮l of tebufenozide during each of the 12 CS⫹ trials across the six concentrations is depicted in Fig. 6. The CS⫺ curves are not shown, but they are consistent with published research (Abramson et al. 1997). Statistically signiÞcant differences were obtained between CS⫹ responses and CS⫺ responses for each of the six insecticide concentrations whether the animals were pretreated with tebufenozide or received it in the US (Table 2). At Þrst glance, such a pattern of results indicates that consuming tebufenozide does not inßuence the discrimination learning of honey bees.

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Fig. 5. Mean proportion of bees responding to 12 CS⫹ trials in a discrimination experiment after a 10-␮l feeding of tebufenozide. For ease of presentation, CS⫺ curves are not presented.

However, an analysis of the data in Fig. 5 shows signiÞcant differences in CS⫹ responding when the concentrations are presented 10 min before discrimination training (F ⫽ 2.841; df ⫽ 1, 150; P ⬍ 0.05). As Fig. 5 shows, although the animals are still able to discriminate, the level of CS⫹ responding is quite scattered and variable. Such performance is in stark contrast to the simple Pavlovian learning situation

studied in experiment 1. There was no signiÞcant concentration ⫻ trial interaction (F ⫽ 1.287; df ⫽ 11, 1,650; P ⬎ 0.05). When tebufenozide is presented in the US, discrimination is more stable. Figure 6 shows that differences in CS⫹ responding among the six concentrations are not as scattered as those in Fig. 5. Analysis of the data in Fig. 6 revealed no signiÞcant differences among the

Fig. 6. Mean proportion of bees responding to 12 CS⫹ trials in a discrimination experiment. The US contained 1 ␮l of tebufenozide. For ease of presentation, CS⫺ curves are not presented.

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Fig. 7. Mean proportion of bees responding to 12 CS⫹ trials in a discrimination experiment after a 10-␮l feeding of dißubenzuron. For ease of presentation, CS⫺ curves are not presented.

concentrations (F ⫽ 2.227; df ⫽ 1, 150; P ⬍ 0.05) but a signiÞcant concentration ⫻ trial interaction (F ⫽ 1.501; df ⫽ 11, 1,650; P ⬍ 0.05). Diflubenzuron. Fig. 7 shows CS⫹ responding in honey bees pretreated with 10 ␮l of dißubenzuron across the six concentrations. The performance of animals receiving 1 ␮l of dißubenzuron during each of the 12 CS⫹ trials across the six concentrations is presented in Fig. 8. The CS⫺ are not shown.

Statistically signiÞcant differences were obtained between CS⫹ responses and CS⫺ responses for each of the six insecticide concentrations whether the animals were pretreated with dißubenzuron or received it in the US (Table 2). At Þrst glance, such a pattern of results suggests that consuming dißubenzuron does not inßuence the ability of honey bees to discriminate between two CSs, one of which is consistently followed by a US.

Fig. 8. Mean proportion of bees responding to 12 CS⫹ trials in a discrimination experiment. The US contained 1 ␮l of dißubenzuron. For ease of presentation, CS⫺ curves are not presented.

April 2004


The CS⫹ curves in Fig. 7 show an acceptable level of CS⫹ responding. Analysis of the data in Fig. 7 reveals no signiÞcant differences in CS⫹ responding when any of the six dißubenzuron concentrations are prefed to the honey bees (F ⫽ 0.704; df ⫽ 1, 150; P ⬎ 0.05). There was a signiÞcant concentration ⫻ trial interaction (F ⫽ 1.491; df ⫽ 11, 1,650; P ⬍ 0.05). Interestingly, the situation is very different in animals receiving 1 ␮l of dißubenzuron during each of the 12 CS⫹ trials. Figure 8 shows that CS⫹ responding is very poor. Animals receiving 0.55 liter/ha, for example, seem to begin acquiring the discrimination around the third CS⫹ trial. As training progresses the number of CS⫹ responses actually decreases. Although the CS⫹ responses are low, the CS⫺ responses for the 0.55 liter/ha group are signiÞcantly lower. There were no signiÞcant differences of concentration (F ⫽ 0.951; df ⫽ 1, 150; P ⬎ 0.05), nor was there a signiÞcant concentration ⫻ trial interaction (F ⫽ 1.167; df ⫽ 11, 1,560; P ⬎ 0.05). An examination of Table 2 shows that, although there were signiÞcant differences between CS⫹ and CS⫺ responding in all groups receiving dißubenzuron, the concentration ⫻ trial interaction is signiÞcant only for animals receiving 1 ␮l of sucrose (no insecticide). We note that the dißubenzuron complex learning results may be viewed with caution. This is suggested because of the relatively low levels of CS⫹ responding in animals receiving sucrose only. We expected to Þnd higher levels of discrimination learning. Although every effort was made to control for calendar variables, there are times during the bee running season that animals are difÞcult to condition. There was no a priori reason to discard any animal and we assume that such effects are equally distributed among the bees selected for each dayÕs experiments. Discussion In the simple learning experiments where bees were trained to acquire an association between the odor of cinnamon and feeding, and the complex learning experiments where bees were required to learn a discrimination, all experimental animals performed signiÞcantly better than their controls. This difference occurred in both a group design by using paired versus unpaired animals and in a single-subject design in which an animal was trained to discriminate between two CSs. Such differences between experimental subjects and control bees suggest that exposure to tebufenozide and dißubenzuron does not inßuence learning of harnessed honey bee foragers. However, an inspection of the learning curves revealed a subtle effect of insecticides on learned behavior. We preface our discussion on the inßuence of tebufenozide and dißubenzuron on learning by pointing out that we do not know whether learning is affected directly or is inßuenced by a disruption in some secondary system such as the feeding reßexes, and/or sensory systems responsible for olfaction and taste. The problem of interpretation is compounded by the fact that tebufenozide and dißubenzuron have

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different modes of action and that different concentrations were used for the two insecticides. Nevertheless, we are comfortable making several generalizations. In the simple learning experiments, pretreating bees with 10 ␮l of tebufenozide, 10 min before training, produced lower levels of acquisition when the concentrations were 0.22, 0.29, and 1.0 liter/ha. Although the levels of acquisition were lower, the performance of paired versus unpaired animals was still statistically signiÞcant. A similar, although less pronounced effect was also seen in animals pretreated with dißubenzuron. Acquisition was especially poor in animals pretreated with a 0.55 liter/ha concentration of dißubenzuron, where approximately one-half the bees responded to the CS. The ability of honey bees to learn a simple association between a CS and US was also inßuenced when bees received concentrations imbedded in the US. When animals were given a series of 1-␮l droplets of dißubenzuron over the course of 12 training trials, the effects on acquisition and extinction were striking. After a period of initial acquisition, performance declined over the remaining training trials and remained so during extinction. When tebufenozide was used during the course of training, acquisition was not signiÞcantly different from untreated bees given a series of 1-␮l droplets of sucrose. This suggests that honey bees are affected more by exposure to dißubenzuron than to tebufenozide when learning a simple Pavlovian conditioning task. Perhaps the greatest effect of tebufenozide and dißubenzuron was on discrimination learning. For this type of learning, bees are required to discriminate between two CSs, with one always paired with a US. Despite statistically signiÞcant differences between CS⫹ and CS⫺, the acquisition curves reveal the subtle effect these IGRs have on honey bee learning. When animals were pretreated with 10 ␮l of tebufenozide, CS⫹ responding was relatively poor with little evidence of an acquisition effect. The results were better when animals received a series of 1-␮l droplets. Even here, however, ⬇50% of the animals receiving a 1.0 liter/ha concentration respond to the CS⫹, compared with 80% of the animals receiving a series of 1-␮l droplets of sucrose. Interestingly, the results with dißubenzuron were in the opposite direction. Discrimination performance was not affected when animals were pretreated with 10 ␮l of dißubenzuron but was signiÞcantly affected when the animals received a series of 1-␮l droplets. Honey bees receiving a concentration of 0.55 liter/ha seldom responded to the CS⫹. A statistically signiÞcant difference between the CS⫹ and CS⫺ occurred only because animals seldom responded to the CS⫺. This suggests discrimination learning is also affected in honey bees by exposure to dißubenzuron and tebufenozide. Our results point toward the need to design learning experiments examining the effect of agrochemicals on honey bee behavior. The results also provide further support for the view that the proboscis extension reßex is an effective bioassay for insecticide assessment

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(Decourtye and Pham-Dele` gue 2002). Our method of studying both acquisition and extinction and simple and complex learning seems to be a good strategy to detect differences in performance when evaluating agrochemicals (Stone et al. 1997, Weick and Thorn 2002). Existing behavioral tests have primarily focused on sublethal effects of insecticides. The results presented here suggest that effort be directed toward using learning paradigms to study the new generation synthetic pyrethroids, additional IGRs, and fermentation by-products. We believe the learning paradigm is a good place to start (Abramson 1994, 1997). Designs are available to measure higher order processes such as compound conditioning, short-term memory, and latent inhibition in both harnessed and free-ßying animals (Smith and Abramson 2003). Moreover, it is also possible to place the insecticide in compound with the scent used as the conditioned stimulus. This manipulation can be included to determine whether any of the insecticides are repellent. If so, this would be expected to interfere with the development of a learned association between landing on a ßower and feeding, thereby reducing the effect of the insecticide on learning (Abramson et al. 1999). We hope the results presented here will stimulate the use of learning paradigms to evaluate agrochemicals considered “safe” for honey bees and other insect pollinators. Acknowledgments We thank Brenda Morales and Noe Ramos whose participation was made possible by NSF-REU grant no. SES-0097643. Thanks are also due to Darius Donohue, Robbie Coffelt, Jaci Tennies, Josh Gibson, Kori McMillian, Laci Harp, April Williams, Lynn Michaluk, Cassie Blair, Brooke Brunken, Stephanie Willingham, Blaine Browne, and Noah Sears, who assisted in the experiments (all were students in the Department of Psychology, Oklahoma State University). This study was supported by a grant from the Oklahoma Center for Water Research. SquireÕs participation was made possible by an exchange program between Oklahoma State University and the University of Hertfordshire, United Kingdom.

References Cited Abramson, C. I. 1994. A primer of invertebrate learning: the behavioral perspective. American Psychological Association, Washington, DC. Abramson, C. I. 1997. Where have I heard it all before: some neglected issues of invertebrate learning, pp. 55Ð78. In G. Greenberg and E. Tobach [eds.], Comparative psychology of invertebrates: the Þeld and laboratory study of insect behavior. Garland Publishing, New York.

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Abramson, C. I., D. A. Buckbee, S. Edwards, and K. Bowe. 1996. A demonstration of virtual reality in free-ßying honey bees: Apis mellifera. Physiol. Behav. 59: 39 Ð 43. Abramson, C. I., M. C. Silva, and J. M. Price. 1997. Learning in the Africanized honey bee and Apis mellifera. Physiol. Behav. 62: 657Ð 674. Abramson, C. I., I. S. Aquino, F. S. Ramalho, and J. M. Price. 1999. Effect of insecticides on learning in the Africanized honey bee (Apis mellifera L.). Arch. Environ. Contamin. Toxicol. 37: 529 Ð535. Decourtye, A., and M. H. Pham-Dele`gue. 2002. The proboscis extension response: assessing the sublethal effects of insecticides on the honey bee, pp. 67Ð 84. In J. Devillers and M. H. Pham-Dele` gue [eds.], Honey bees: estimating the environmental impact of chemicals. Taylor & Francis, Ltd., London, England. Dhadialla, T. S., G. R. Carlson, and D. P. Le. 1998. New insecticides with ecdysteroidal and juvenile hormone activity. Annu. Rev. Entomol. 43: 545Ð569. Heller, J., H. Mattioda, E. Klein, and A. Sagenmuller. 1992. Field evaluation of RH 5992 on lepidopterous pests in Europe. Brighton Crop Prot. Conf. Pests Dis. 1: 59 Ð 65. Information Ventures, Inc. 1995. Dißubenzuron pesticide fact sheet. http://infoventures.com/e-hlth/pesticide/ dißuben.html. Mamood, A. N., and G. D. Waller. 1990. Recovery of learning responses by honeybees following a sublethal exposure to permethrin. Physiol. Entomol. 15: 55Ð 60. Smith, B. H., and C. I. Abramson. 2003. Case studies in insect behavior, pp. 258 Ð263. In J. H. Byrne, H. Eichenbaum, H. Ruediger, III, and R. F. Thompson [eds.], Encyclopedia of learning and memory, 2nd ed. Macmillian, New York. Stone, J. C., C. I. Abramson, and J. M. Price. 1997. Task dependent effects of dicofol (Kelthane) on learning in the honey bee (Apis mellifera). Bull. Environ. Contamin. Toxicol. 58: 177Ð183. Taylor, K. S., G. D. Waller, and L. A. Crowder. 1987. Impairment of a classical conditioning response of the honey bee (Apis mellifera L.) by sublethal doses of synthetic pyrethroid insecticides. Apidologie 18: 243Ð252. Thompson, H. M. 2003. Behavioural effects of pesticides in bees Ð their potential for use in risk assessment. Ecotoxicology 12: 317Ð330. Tripathi, R. 1996. Information sheet on Dimilin. Bureau of Toxic Substances, Virginia Department of Health, Richmond, VA. Walgenbach, J. F. 1999. Attributes of ConÞrm and Spintor. North Carolina Cooperative Extension Service. Apple Insect News July 13 (http://ßetcher.ces.state.nc.us/ staff/cpalmer/july991.html). Weick, J., and R. S. Thorn. 2002. Effects of acute sublethal exposure to coumaphos or diazinon on acquisition and discrimination of odor stimuli in the honey bee (Hymenoptera: Apidae). J. Econ. Entomol. 9: 227Ð236. Received 23 June 2003; accepted 23 December 2003.