Biogenic Amine Levels Change in the Brains of Stressed Honeybees

Transcription

1 Archives of Insect Biochemistry and Physiology 68: (2008) Biogenic Amine Levels Change in the Brains of Stressed Honeybees Yi-Ling Chen, 1 Yu-Shan Hung, 1 and En-Cheng Yang 1,2 * Three different stress treatments, CO 2 anesthesia, chilling anesthesia, and vertical spin, were applied to test whether honeybee (Apis mellifera) workers express stress responses in rewarding behaviors. In the present work, we defined the rewarding behaviors as the bees flying between the hive and feeder. The results from behavioral observation show that the flight time interval of the rewarding behavior of bee workers, flying between hive and feeder, was longer when they were stressed, suggesting that the stress treatments affected the workers rewarding behavior. The biogenic amine levels in the workers brains were measured to examine the rapid biochemical brain response to the stressors. After the chilling anesthesia, the dopamine (DA) and octopamine (OA) levels were significantly decreased; with the CO 2 anesthesia for durations of both 2 min and 4 min, only DA showed a significant decrease. In the non-anesthesia treatments, the vertical spin with a velocity of 50 and 60 rpm for 90 s, the DA and OA levels were significantly decreased. Our results suggest that when the bees were under stress, the brain levels of OA and DA were depressed, and this may have caused latency in the rewarding behavior. The serotonin (5-HT) levels under these stress treatments were not changed. Arch. Insect Biochem. Physiol. 68: , Wiley-Liss, Inc. KEYWORDS: stress physiology; biogenic amine; rewarding behaviors; honeybee INTRODUCTION Biogenic amines play a number of roles in regulating various physiologies and in producing behaviors in insects. High concentrations of octopamine (OA), dopamine (DA), and serotonin (5-HT) exist in the insect nervous system (Evans, 1980; Mercer, 1987), and that these biogenic amines are the common neurotransmitters, neuromodulators and neurohormones in invertebrates (Evans, 1980; Brown and Nestler; 1985; Roeder, 1994). Recent studies have demonstrated that the levels of these biogenic amines changed under unfavorable conditions in various insect species. The change of biogenic amine levels caused by stressors were found in high or low temperature (Hirashima et al., 2000; Chentsova et al., 2002), mechanical or chemical stimuli (Harris et al., 1996), high population density (Kozánek et al., 1986), and handling stress caused by grasping a bee s leg (Harris and Woodring, 1992). After heat treatment, the biogenic amines, particularly the OA levels, changed significantly in Drosophila virilis, Schistocerca gregaria, and Periplaneta american (Davenport and Evans, 1984; Hirashima et al., 2000). The harmful effects of anesthesia by chilling or CO 2, on copulation behavior of the fruit fly, D. melanogaster, were recently reported. After recovering from the anesthesia, copulation latency was significantly increased (Barron, 2000). The effects of CO 2 anesthesia on the behaviors and changes in 1 Department of Entomology, National Chung Hsing University, Taichung, Taiwan 2 Department of Entomology, National Taiwan University, Taipei, Taiwan Grant sponsor: National Science Council, Taiwan; Grant number: NSC B *Correspondence to: En-Cheng Yang, Department of Entomology, Laboratory of Insect Neurobiology, National Taiwan University, Taipei 10617, Taiwan. [email protected] 2008 Wiley-Liss, Inc. DOI: /arch Published online in Wiley InterScience (

2 242 Chen et al. biogenic amine levels in hemolymph of honeybees has also been reported (Harris et al., 1996; Pankiw and Page Jr., 2003). Potent reciprocal relationships exist between biogenic amines and behavior. Four biogenic amines, OA, DA, 5-HT, and norepinephrine (NA), have been detected in different parts of the honeybee brain (Schürmann et al., 1989; Brandes et al., 1990; Harris and Woodring, 1992, 1995; Taylor et al., 1992; Kreissl et al., 1994). The cerebral ganglion contains more DA than OA, approximately the same levels of DA and 5-HT, and very low levels of NA (Mercer et al., 1983). Amines levels in the brain vary with age (Fuchs et al., 1989; Harris and Woodring, 1992) and behavioral state (Taylor et al., 1992), suggesting that the amines may be involved in controlling the complex changes in behavior that underlie colony division of labor (Schulz and Robinson, 2001; Barron et al., 2002; Schulz et al., 2002). In addition, biogenic amines are significantly involved in modulating learning and memory in the bee brain. Application of amines to a bee s brain affects sensitivity to olfactory stimuli and learning performance (Macmillan and Mercer, 1987; Michelsen, 1988; Menzel et al., 1988). The mushroom body in the honeybee brain is the key part in the insect s memory and learning system; global injections of DA or OA into a bee s brain demonstrate that these amines are general facilitating modulators for memory storage and retrieval, whereas 5-HT inhibits the retrieval of memory by antagonizing the action of OA. On the other hand, DA selectively affects the memory processes when injected into the median part of the brain (close to or in the central complex) (Menzel et al., 1989). In addition, under various conditions, e.g., different ages, different seasons, different colonies, or grabbing the bee s leg with forceps, the brain levels of biogenic amines are also significantly changed (Harris and Woodring, 1992). Based on this evidence, we posed the hypothesis that rewarding behavior and brain biogenic amines change in honeybees under stress. Moreover, in the foraging process, a forager memorizes the location of various nectar sources and establishes a stable foraging pathway between her hive and these feeding places (Free, 1963; Waddington, 1983). Foraging bouts (going back and forth between the hive and feeding places) could be related to mid/long-term memory (Menzel, 1999). A bee s long-term memory guides foraging behavior and can be separated from short-term memory (Frisch, 1967; Menzel, 1990, 1999). Therefore, if a bee forager is stressed, our hypothesis predicts the bee s rewarding behavior, related to long-term memory, will change? Here, we show that flight time interval of the rewarding behavior of bees was longer than usual after they were stressed by artificial stressors, and the biogenic amines levels of brain revealed significant changes. MATERIALS AND METHODS Animals Nine colonies of honeybees (Apis mellifera) maintained with normal beekeeping techniques, either in the university farm or on the campus was used for both the behavioral and physiological experiments. Behavioral Tests A rewarding pathway was established by attracting bees, with a feeder to an experimental station, 70 m away from the beehives. A fresh aqueous solution of sucrose and honey, concentrated just enough to keep bees coming regularly (50%, w/ v), was placed in the feeder. The bees needed at least 1 2 days to establish the rewarding pathway. All behavioral experiments were carried out at this experimental station from 10:00 to 17:30 on sunny days during the summer season (May August, 2002). Ten randomly chosen foragers were individually marked with colors, painted on the dorsal surface of the thorax, before beginning the experiments. The flight time intervals of marked bees, rewarded continuously 10 times, were recorded. After the 10 rewards, each of the bees was gently induced into a plastic test tube (ID = 16 mm, L = 100 mm, V = 15 ml), which was then capped for chilling anesthesia or vertical spin, or placed into

3 Biogenic Amines Level in Stressed Bee Brain 243 a plastic bag for CO 2 anesthesia. The motionless bees that had been treated with chilling or CO 2 anesthesia, were released on a dish (diameter = mm) near the feeders to recover. The flight time interval between when the bees left and returned to the experimental station was recorded as the response of treatment. Following the response of treatment, the flight time intervals of a further 10 rewards were recorded. Application of Stressors To be sure that the flight time interval between when the bees left and returned to the feeder was affected by stress, 10 bees were anesthetized with chilling treatment for a pilot study. The bees were captured in a test tube, and then the whole test tube was immersed in ice. The bees were immobile after 1 min and were chilled for another 3 min. After this chilling anesthesia, the bees generally needed more than 30 s to recover; when fully recovered, they flew away from the experimental station as they normally did. To anesthetize the bees, while avoiding the effects of low temperatures, the CO 2 anesthesia treatment was applied by introducing the bees into a CO 2 -filled plastic bag. The bees were immobilized within 5 s. Ten of the CO 2 -anesthetized bees were kept in the CO 2 -filled bag for 2 min, while another 10 bees were kept in the bag for 4 min. The CO 2 anesthetized bees recovered in the same manner as those exposed to the chilling treatment. Bee stress, without anesthesia, was induced by a vertical spin. Ten test tubes, each with one marked bee inside, were inserted into the rotor of an Orbital Shaker (Firstek Scientific; T-101; spin radius = 15 cm). Four experiments with spin rotations at 30, 40, 50, and 60 rpm were conducted to test the effects of the velocity. The durations for each of the four spin velocities were 30, 60, and 90 s. After the spinning treatment, the bees were released near the feeders at the experimental station. Since the bees were not anesthetized, they flew away directly from the station. A group of 10 marked bees were kept in test tubes for 30, 60, and 90 s, without spinning treatment, as the control. All the bees recovered after being stressed, and no mortalities were recorded during the experiments. In order to measure the biogenic amine levels in the brain, another 20 bee heads were cut off and frozen with liquid nitrogen immediately, when recovering from chilling or CO 2 anesthesia treatment but not yet fly away. The control groups bees were gathered as they were feeding and their heads were removed. Immediately after the vertical spin treatments, 20 bee heads were cut off by scissors. In this case, the heads of the control bees were removed after the bees had been kept in the test tubes for 90 s. All the heads were stored frozen in liquid nitrogen until the brains were isolated for analysis. Reagents and Instrumentation OA, DA, and 5-HT were obtained from Sigma Chemical Co. (St. Louis, MO); ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) was obtained from Fluka Chemika (Buchs, SUI); methanol and trifluoroacetic acid (TFA) were obtained from Mallinckrodt Baker (Phillipsburg, NJ, USA). Water from a Milli-Q purification system was used to make all solutions and dialysis filters. The high intensity ultrasonic processor (Vibra-Cell, Sonics & Materials, Newtown, CT, USA) was used for brain sample preparation. The high-performance liquid chromatography (HPLC) system comprised a SpectraSYSTEM P1000 isocratic pumps, solvent degassers (Spectra-Physics Analytical, Fremont, CA), and SSI 502 fluorodetector (Scientific Systems, State College, PA, USA). Data were processed on an IBM-PC compatible computer using the LabVIEW program (version 6.0, National Instruments, Austin, TX) and Origin data system software (version 7.0, OriginLab, Northampton, MA). Standard and Sample Preparation The standard and sample preparation procedures followed established methods (Harris and Woodring, 1992; Wood and Hall, 2000). Fresh HPLC standards were prepared each day by taking aliquots from stock solutions and diluting them

4 244 Chen et al. to various concentrations with water (OA: µm; DA: µm; 5-HT: µm). The brains were removed from bee heads that stored in liquid nitrogen before dissection. Brains (20/sample) were combined with 140 µl of icecold 2 % EDTA and homogenized with pellet pestle in the 1.5-ml Eppendorf tubes. Each sample was then sonicated for min and centrifuged at 13,000 rpm (15,000g) for 10 min at 4 C. The supernatant was transferred to a 0.22-µm nylon membrane filter in a new Eppendorf tube. The filtrates were then frozen (in liquid nitrogen) until needed. Just before analysis, filtrates were thawed and injected directly onto the HPLC column. Biogenic Amines Detection and Quantization Biogenic amines were assayed using the reversed-phase HPLC procedure of Wood and Hall (2000) as previously described. Standards and samples were analyzed at room temperature using 5µm Thermo Hypersil-Keystone BDS C18 ( mm ID) column (Thermo, Tasmania, Australia) to separate. Mobile phase A consisted of 0.05% aqueous trifluoroacetic acid (TFA)-methanol (97.5: 2.5, v/v) and mobile phase B consisted of 0.05% aqueous TFA-methanol (40:60, v/v). The mobile phases were prepared as needed and degassed by helium sparging before use. Injection volumes of both standards and brain EDTA samples were 10 µl. A flow rate of 1.0 ml min was used over 20 min. Biogenic amines detection was by fluorodetector using excitation and emission wavelengths set at 220 nm at 320 nm, respectively. Biogenic amines were identified and quantified by comparing peak areas with known standards. For precise identification the biogenic amine peak, the two methods of either adding three biogenic amine slanderers into the sample, and/or changing the flow rate, were employed to confirm peak identities. The signals from our detector were captured by a multifunction data acquisition system (model NI 435x, National Instruments, Austin, TX) (Notch Filter Freq = Hz), via the computer. The signals could, therefore, be monitored on the PC display and processed by the LabVIEW program (version 6.0, National Instruments). The peak areas of detected signals were quantified with Origin data system software (version 7.0, OriginLab). Statistical Analysis In the chilling and CO 2 anesthesia treatments, statistical analyses for behavior were expressed as the flight time interval: mean (min) ± standard error (SE) and performed in Student s t-test. In the vertical spin treatments, the treatment responses were analyzed using two-way analysis of variance (ANOVA) in SAS (version 8.01, SAS Institute, Cary, NC). Differences in the three time durations of the various spin velocities were then determined by ANOVA in SAS. The multiple comparisons following ANOVA were analyzed by Tukey s Studentized Range Test in SAS. For HPLC analysis of biogenic amines, statistical comparisons of brain levels were performed by Student s t-test. In all cases, P < 0.05 was considered statistically significant. RESULTS Effects of Stressors on Rewarding Behavior Chilling anesthesia. After the chilling treatment, the bees recovered from anesthesia in 1 min. The recovering bees first gasped their abdomens and then swung their legs. These movements seemed clumsy. Subsequent to these behaviors, the bees stood up gradually, as they normally did, and frequently cleaned and scratched their compound eyes and antennae until eventually flying away from the experimental station. They were observed as fully recovered for long periods from 10 to 60 min, and were buzzing around the experimental station with no obvious abnormal behaviors. Before the chilling anesthesia, the average flight time interval of rewards for the 10 marked bees (pre-treatment response) was 2.1 ± 0.2 min. After the first chilling anesthesia, the average flight time interval of the treatment response between when the bees left and returned to the experimental station was 44.9 ± 6.2 min. Then the average flight time interval of rewards (post-treatment response)

5 Biogenic Amines Level in Stressed Bee Brain 245 TABLE 1. Effects of Chilling and CO 2 Anesthesia Treatments on Honeybee Rewarding Behaviors (Flight Time Intervals) (P < 0.05)* Mean (min) ± SE Treatments N Pre-treatment Treatment response Post-treatment Retreatment response Post-retreatment Chilling anesthesia ± 0.2 a 44.9 ± 6.2 b 2.7 ± 0.6 a 63.5 ± 10.3 b 3.8 ± 0.6 a CO 2 anesthesia 2 min ± 0.6 a ± 22.2 b 10.6 ± 5.0 a 4 min ± 0.5 a 38.7 ± 8.8 b 2.6 ± 0.4 a *Pre-treatment: before treatment, the average flight time intervals of bees rewarded continuously 10 times; treatment response: after first treatment, the average flight time interval of the treatment response between the bees leaving and returning to the experimental station; post-treatment: the average flight time intervals of bees rewarded continuously 10 times after first treatment; retreatment response: after the continuous second treatment, the average flight time interval of the treatment response between the bees leaving and returning to the experimental station; post-retreatment: the average flight time intervals of bees rewarded continuously 10 times after second treatment. The values having the same letter are not significantly different. was about 2.7 ± 0.6 min after the first chilling anesthesia (Table 1). The treatment response was significantly different from both the pre-treatment and post-treatment responses (both P < 0.01). To test whether another chilling anesthesia would cause the same or even stronger effects in rewarding behavior, a second chilling treatment was applied on these chill-experienced bees. The average flight time intervals of the second chilling anesthesia response (retreatment response) were 63.5 ± 10.3 min, which is significantly different from the average flight time interval following rewards (post-retreatment: 3.8 ± 0.6min; P < 0.01) (Table 1). Even though these bees were anesthetized twice with chilling anesthesia, they still performed normally in their post-treatment rewarding behavior. Both the first and second chilling treatment significantly increased the flight time interval latency, after the anesthetized bees had fully recovered from the anesthesia and flew away from the experimental station as normal. This result also demonstrates that the flight time interval latency corresponded with the chilling treatment. CO 2 anesthesia. The behaviors of the bees recovering from CO 2 anesthesia were the same as for the chilling anesthesia described above, although the bees needed more time to fully recover from the CO 2 anesthesia. Before the CO 2 anesthesia, the average flight time interval of the 9 marked bees (one was lost during the experiment) rewards (pretreatment) was 3.0 ± 0.6 min, whereas the response of the 2 min CO 2 treatment was ± 22.2 min and the post-treatment was 10.6 ± 5.0 min (Table 1). Treatment response comparing pretreatment and post-treatment results were significantly different (P < 0.01; P < 0.01). In another CO 2 anesthesia, when the bees were treated with CO 2 for 4 min, the treatment response was 38.7 ± 8.8 min, which was also significantly different from their pre- and post-treatment intervals (pre-treatment = 3.3 ± 0.5 min, P < 0.01; post-treatment = 2.6 ± 0.4 min, P < 0.01) (Table 1). The CO 2 treatments of 2 min and 4 min demonstrated that the reward behavior of bees is also influenced by CO 2 anesthesia. Stress induced by vertical spin. After all the spinning treatments, the treated-bees were released near the feeders and most of the bees rapidly flew away from the station. In the treatments, four experiments were conducted with spin velocities of 30, 40, 50, and 60 rpm, the durations for each of the four spin velocities being 30, 60, and 90 s. The main trend was that the 40, 50, and 60 rpm treatments for 90 s had more significant treatment responses than the 30 rpm or control treatments (Table 2). Therefore, the rewarding behaviors of bees were indeed individually influenced by vertical spinning under 40, 50 and 60 rpm for 90 s duration. TABLE 2. Multiple Comparisons From Tukey s Studentized Range (HSD) Test analyzing the Effects of Various Spin Velocities for 90 s on Honeybee Rewarding Behaviors* Velocity (rpm) Duration (s) n Treatment response; Mean (min) ± SE Control ± 0.5 a ± 0.4 a ± 1.0 b ± 1.4 b ± 1.9 b *Treatment response: after vertical spin treatment, the average flight time interval of the treatment response between the bees leaving and returning to the experimental station. The values having the same letter are not significantly different.

6 246 Chen et al. Stressor-Induced Changes in Biogenic Amine Levels Effects of chilling anesthesia on biogenic amine levels. Besides the abnormal rewarding behavior, the brain levels of OA (270.6 ± pg/brain, P = 0.02) and DA (329.5 ± pg/brain, P = 0.002) were significantly lower after the chilling anesthesia treatment, when compared to the control (OA: ± pg/brain; DA: ± pg/brain) (Fig. 1A, D). The brain level of 5-HT was not changed, however (Fig. 1G). The decrease of biogenic amine levels in the bees may be attributed to the effects of the chilling anesthesia treatment. Effects of CO 2 anesthesia on biogenic amine levels. Brain levels of DA were significantly lower when bees were treated with CO 2 for 2 min or 4 min (2 Fig. 1. Effects of stressors on biogenic amines levels in the brains of Apis mellifera. Levels of octopamine (A), dopamine (D), and serotonin (G) with chilling anesthesia applied; levels of octopamine (B), dopamine (E), and serotonin (H) with CO 2 anesthesia for durations of 2 and 4 min applied; the levels of octopamine (C), dopamine (F) and serotonin (I) with different vertical spin velocity for 90 s applied. Each amine column represents the average (mean ± SE) of 3 samples (20 brains/sample). Groups that differed significantly from one another in levels of biogenic amines are indicated with different letters above the bars (P < 0.05); statistical comparisons were performed by Student s t-test.

7 Biogenic Amines Level in Stressed Bee Brain 247 min: ± pg/brain, P = 0.01; 4 min: ± pg/brain, P < 0.01) (Fig. 1E). In contrast to DA, both OA and 5-HT levels did not change with these treatments (Fig. 1B,H). No significant difference was found in the levels of these biogenic amines between the 2-min and 4-min CO 2 treatments. This result demonstrated that the biogenic amines in the bees brains were affected by 2-min and 4-min CO 2 anesthesia; in other words, the DA level was lowered by both CO 2 treatments of 2 and 4 min, while there was no effect found in the OA and 5-HT levels. Effects of different vertical spin velocity on biogenic amine levels. After vertical spin treatments (40, 50, and 60 rpm for 90 s), the biogenic amine levels of bee brain were measured. In these experiments, a control (bee kept in test tube for 90 s without spinning) was applied. The treatments with different spin velocities affected significant changes in OA and DA levels of bee brains (Fig. 1C,F). In OA levels, the treatment at 50 rpm showed a significant difference from the control (P = 0.044). The control (P < 0.01), 50 rpm (P = 0.031) and 60- rpm spin treatments (P = 0.015) showed significant differences from the free bees groups (Fig. 1C). The main trend was that for rising vertical spin velocities (40, 50, and 60 rpm), the OA levels showed decreasing tendencies (Fig. 1C). In DA levels, only the 60-rpm vertical spin treatment showed a significant difference from the control (P = 0.012) (Fig. 1F). The control (P < 0.01), 40- rpm (P < 0.01), 50-rpm (P < 0.01) and 60-rpm treatments (P < 0.01) showed significant differences from the free bees groups (Fig. 1F). These results demonstrated that the changes in OA and DA levels in bees brains were affected by both the control and by different vertical spin velocities; in addition, no significant changes occurred in 5-HT levels (Fig. 1I). DISCUSSION Stress Behaviors The honeybee foragers displayed significantly increasing flight time intervals for rewarding behavior after the stress treatments. In both the chilling and CO 2 anesthesia treatments, the flight time delay intervals were 10 times greater than those found for spin treatment responses, suggesting that the anesthesia had a more detrimental effect on the foragers than the non-anesthesia treatment. The bees also needed a longer period to recover and frequently cleaned their compound eyes and antennae when they awoke from the anesthesia, as well as not going directly back to the hive after they left the experimental station. The bees treated with vertical spinning expressed longer return latency and also stopped on the way to hive to clean their compound eyes and antennae. This particular stress behavior is similar to recent observations of bees poisoned in the field (Schmuck et al., 2001). In spite of the short-term changes in rewarding behavior, some long-term effects from the chilling or CO 2 anesthesia were also observed. Barron (2000) has demonstrated that both chilling and CO 2 anesthesia significantly increased the copulation latency of flies, even though the flies were given 20 h to recover from the anesthesia. Anesthesia also disrupts some forms of memory (Tully et al., 1990, 1994; Tully, 1996). Thus, it has been suggested that more careful consideration should be given to the effect anesthetic has on animals and that handling stresses should be reduced whenever it is necessary to perform behavioral or physiological experiments (Barron, 2000). Changes in Biogenic Amine Levels Under Stress In physiology, biogenic amines play an important role the learning and memory of bees (Mercer and Menzel, 1982). Global injections of amines into to bee brains modulate memory formation and retrieval in a dose dependent fashion; biogenic amines, OA, DA, and 5-HT, are involved in the formation and retrieval of olfactory memory. The storage and retrieval of memory function is facilitated when OA is micro-injected into the calyces of the mushroom body in the honeybee brain; in contrast, memory storage was retarded and retrieval was reduced when 5-HT was micro-injected into the same location (i.e., this antagonized the action of OA).

8 248 Chen et al. DA, however, selectively affects the retrieval processes when injected into the median part of the brain (close to or into the central complex). By analyzing the brain levels of OA, DA, and 5-HT, this study has found that during anesthesia, the DA and OA (only in chilling anesthesia) levels were significantly reduced. In a vertical spin, without anesthesia, the DA level was also significantly reduced and the OA level showed a decreasing tendency as the spinning velocity increased. These findings, together with previous studies on the effects of learning and memory, show that the changes in the levels of biogenic amines in bees brains may be related to the application of stressors as well as their effects on stress physiology. Since DA affects the retrieval processes of a bee s learning and memory (Mercer and Menzel, 1982), the effects stress has on DA levels may be related to temporary long-term memory loss, and may therefore lead to abnormal rewarding behavior, significantly increasing the flight time intervals. Bees quickly and effectively learn local cues from flowers (color, odor, shape, location, handling), according to the reward provided (nectar, pollen). Menzel (1999) first proposed that the formation of a bee s foraging cycle is a time-consuming process, which depends on the interval between sequential learning trials. A foraging bout is structured in time, over several hours or months, to form mid/long-term memory. In the present study, the flight time interval increased after chilling, CO 2 anesthesia and some specific spin treatments, suggesting that the memories of the bees were temporarily impaired. If so, the mid/longterm memory of the bees was impaired temporarily by the stressors. It has been reported that a brief cooling treatment (5 or 10 s) in the calyx area of a bee s mushroom body resulted in significant impairment of memory formation (Erber et al., 1980). Further study on the physiological effects of stressors in the mushroom body of bees brains is warranted. ACKNOWLEDGMENT The authors would like to express our appreciation to the anonymous reviewers for insightful comments and improvements. This work was supported by a grant (NSC B ) from the National Science Council, Taiwan. LITERATURE CITED Barron AB Anaesthetising Drosophila for behavioural studies. J Insect Physiol 46: Barron AB, Schulz DJ, Robinson GE Octopamine modulates responsiveness to foraging-related stimuli in honeybee bees (Apis mellifera). J Comp Physiol A 188: Brandes C, Sugawa M, Menzel R High-performance liquid chromatography (HPLC) measurement of catecholamines in single honeybee brains reveals caste-specific differences between worker bees and queens in Apis mellifera. Comp Biochem Physiol 97C: Brown CS, Nestler C Catecholamines and indolalkylamines. In: Kerkut GA, Gilbert LI, editors. Comprehensive insect physiology: biochemistry and pharmacology. Oxford: Pergamon. p Chentsova NA, Gruntenki NE, Bogomolova EV, Adonyeva NV, Karpova EK, Rauschenbach IY Stress response in Drosophila melanogaster strain inactive with decreased tyramine and octopamine contents. J Comp Physiol B 172: Davenport AP, Evans PD Stress-induced changes in the octopamine levels of insect haemolymph. Insect Biochem 14: Erber J, Masuhr TM, Menzel R Localization of shortterm memory in the brain of the bee, Apis mellifera. Physiol Entomol 5: Evans PD Biogenic amines in the insect nervous system. Adv Insect Physiol 15: Free JB The constancy of honeybees. J Anim Ecol 32: von Frisch K The dance language and orientation of bees. Cambridge: Cambridge University Press. Fuchs E, Dustmann JH, Stadler H, Schürmann FW Neuroactive compounds in the brain of the honeybee during imaginal life. Comp Biochem Physiol 92C:

9 Biogenic Amines Level in Stressed Bee Brain 249 Harris JW, Woodring J Effects of stress, age, season, and source colony on levels of octopamine, dopamine and serotonin in the honeybee (Apis mellifera) brain. J Insect Physiol 38: Harris JW, Woodring J Elevated brain dopamine levels associated with ovary development in queenless worker honeybees (Apis mellifera L.). Comp Biochem Physiol C 111: Harris JW, Woodring J, Harbo JR Effects of carbon dioxide on levels of biogenic amines in the brain of queenless worker and virgin queen honeybees (Apis mellifera). J Apic Res 35: Hirashima A, Sukhanova M Jr, Rauschenbach IY Biogenic amines in Drosophila virilis under stress conditions. Biosci Biotechnol Biochem 64: Kozánek M, Juráni M, Somogyiova E Influence of social stress on monoamine concentration in the central nervous system of the cockroach Nauphoeta cinerea (Blattodea), Acta Entomol Bohemoslov 83: Kreissl S, Eichmüller S, Bicker G, Rapus J, Eckert M Octopamine-like immunoreactivity in the brain and suboesophageal ganglion of the honeybee. J Comp Neurol 348: Macmillan CS, Mercer AR An investigation of the role of dopamine in the antennal lobes of the honeybee, Apis mellifera. J Comp Physiol A 160: Menzel R Learning, memory, and cognition in honeybee. In: Kesner RP, Olton DS, editors. Neurobiology of comparative cognition. Hillsdale, NJ: Lawrence Erlbaum Associates. p Menzel R Memory dynamics in the honeybee. J Comp Physiol A 185: Menzel R, Wittstock S, Sugawa M Chemical codes of learning and memory in honeybee. In: Squire LR, Lindenlaub L, editors. The biology of memory. Stuttgart: Schattauer Verlag. p Menzel R, Michelsen B, Ruffer P, Sugawa M Neuropharmacology of learning and memory in honeybees. In: Hertting G, Spatz HC, editors. Modulation of synaptic transmission and plasticity in nervous systems. New York: Springer. p Mercer AR Biogenic amines and the bee brain. In: Menzel R, Mercer A, editors. Neurobiology and behavior of honeybees. Berlin: Springer-Verlag. p Mercer AR, Menzel R The effects of biogenic amines on conditioned and unconditioned responses to olfactory stimuli in the honeybee Apis mellifera. J Comp Physiol 145: Mercer AR, Mobbs PG, Davenport AP, Evans PD Biogenic amines in the brain of the honeybee, Apis mellifera. Cell Tissue Res 234: Michelsen DB Catecholamines affect storage and retrieval of conditioned odour stimuli in honeybees. Comp Biochem Physiol 91C: Pankiw T, Page RE Jr Effect of pheromones, hormones, and handing on sucrose response thresholds of honeybees (Apis mellifera L.). J Comp Physiol A 189: Roeder T Biogenic amine and their receptors in insects. Comp Biochem Physiol C 107:1 12. Schmuck R, Schöning R, Stork A, Schramel O Risk posed to honeybees (Apis mellifera L, Hymenoptera) by an imidacloprid seed dressing of sunflowers. Pest Manag Sci 57: Schulz DJ, Robinson GE Octopamine influences division of labor in honeybee colonies. J Comp Physiol A 187: Schulz, DJ, Elekonich MM, Robinson GE Biogenic amines in the antennal lobes and the initiation and maintenance of foraging behavior in honeybee. J Neurobiol 54: Schürmann FW, Elekes K, Geffard M Dopamine-like immunoreactivity in the bee brain. Cell Tissue Res 256: Taylor DJ, Robinson GE, Logan BJ, Laverty R, Mercer AR Changes in brain amine levels associated with the morphological and behavioral development of the worker honeybee. J Comp Physiol A 170: Tully T Discovery of genes involved with learning and memory: an experimental synthesis of Hirschian and Benzerian perspectives. Proc Natl Acad Sci USA 93: Tully T, Cambiazo V, Kruse L Memory through meta-

10 250 Chen et al. morphosis in normal and mutant Drosophila. J Neurosci 14: Tully T, Boynton S, Brandes C, Dura JM, Mihalek R, Preat T, Villella A Genetic dissection of memory formation in Drosophila melanogaster. Cold Spring Harbor Symp Quant Biol 55: Waddington KD Floral visitation sequences by bees: models and experiments. In: Jones CE, Little RJ, editors. Handbook of experimental pollination biology. New York: Van Nostrand-Reinhold. p Wood AT, Hall MR Reversed-phase high-performance liquid chromatography of catecholamines and indoleamines using a simple gradient solvent system and native fluorescence detection. J Chromatogr A 744: