Responsiveness to sucrose and habituation of the proboscis extension response in honey bees

In honey bees, complex behaviours such as associative learning correlate with responsiveness to sucrose. In these behaviours, the subjective evaluation of a sucrose stimulus influences the behavioural performance. Habituation is a well-known form of non-associative learning. In bees, the proboscis extension response can be habituated by repeatedly stimulating the antennae with a low sucrose concentration. A high sucrose concentration can dishabituate the response. This study tests whether habituation correlates with responsiveness to sucrose in bees of different behavioural states and in bees which are habituated with different sucrose concentrations. Habituation and dishabituation in newly emerged bees, 5-day-old bees and foragers strongly correlated with responsiveness to sucrose. Bees with high responsiveness to sucrose displayed a lower degree of habituation and showed greater dishabituation than bees with low responsiveness. The degree of habituation and dishabituation also depended on the concentration of the habituation stimulus. These experiments demonstrate for the first time in a non-associative learning paradigm that the subjective strength of a sucrose stimulus determines the behavioural performance. Non-associative learning shares this property with associative learning, which suggests that the two processes might rely on similar neural mechanisms.Abbreviations: GRS Gustatory response score - PER Proboscis extension response     

The parasitic mite <Emphasis Type="Italic">Varroa destructor</Emphasis> affects non-associative learning in honey bee foragers, <Emphasis Type="Italic">Apis mellifera</Emphasis> L.

The parasitic mite Varroa destructor influences flight behavior, orientation and returning success of forager honeybees (Apis mellifera) infested as adults. As impaired orientation toward the nest entrance might be due to deficiency in recognition and responsiveness to stimuli in the environment, we examined effects of V. destructor on sensory responsiveness, non-associative and associative learning of honey bee foragers by using proboscis extension reaction paradigm (PER). Although infested and uninfested workers were initially equally responsive to different concentrations of sugar water, we found differences in non-associative learning. In habituation, PER to repeated sugar stimulation of the antennae occurred faster in infested foragers compared to uninfested foragers. In sensitization, infested foragers showed a lower response to an odor stimulus following sugar stimulation than non-infested foragers. Differences in non-associative paradigms were more pronounced in bees with lower responsiveness to sucrose. In conditioning learning experiments, a significant reduction in proboscis extension response was found 1 min but not 12 min after a single conditioning trial indicating that V. destructor predominantly affects the non-associative components of learning and its underlying neural and molecular processes. Jasna Kralj and Axel Brockmann have contributed equally to this study.     

Sucrose acceptance,discrimination and proboscis responses of honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.) in the field and the laboratory

Laboratory studies in honey bees have shown positive correlations between sucrose responsiveness, division of labour and learning. We tested the relationships between sucrose acceptance and discrimination in the field and responsiveness in the laboratory. Based on acceptance in the field three groups of bees were differentiated: (1) bees that accept sucrose concentrations >10%, (2) bees that accept some but not all of the sucrose concentrations <10% and water, and (3) bees that accept water and all offered sucrose concentrations. Sucrose acceptance can be described in a model in which sucrose- and water-dependent responses interact additively. Responsiveness to sucrose was tested in the same bees in the laboratory by measuring the proboscis extension response (PER). The experiments demonstrated that PER responsiveness is lower than acceptance in the field and that it is not possible to infer from the PER measurements in the laboratory those concentrations the respective bees accepted in the field. Discrimination between sucrose concentrations was tested in three groups of free-flying bees collecting low, intermediate or high concentrations of sucrose. The experiments demonstrated that bees can discriminate between concentrations differences down to 0.2 relative log units. There exist only partial correlations between discrimination, acceptance and PER responsiveness.     

Visual associative learning in restrained honey bees with intact antennae

A restrained honey bee can be trained to extend its proboscis in response to the pairing of an odor with a sucrose reward, a form of olfactory associative learning referred to as the proboscis extension response (PER). Although the ability of flying honey bees to respond to visual cues is well-established, associative visual learning in restrained honey bees has been challenging to demonstrate. Those few groups that have documented vision-based PER have reported that removing the antennae prior to training is a prerequisite for learning. Here we report, for a simple visual learning task, the first successful performance by restrained honey bees with intact antennae. Honey bee foragers were trained on a differential visual association task by pairing the presentation of a blue light with a sucrose reward and leaving the presentation of a green light unrewarded. A negative correlation was found between age of foragers and their performance in the visual PER task. Using the adaptations to the traditional PER task outlined here, future studies can exploit pharmacological and physiological techniques to explore the neural circuit basis of visual learning in the honey bee.     

Effect of pheromones,hormones, and handling on sucrose response thresholds of honey bees (Apis mellifera L.)

The responsiveness of bees to sucrose is an important indicator of honey bee foraging decisions. Correlated with sucrose responsiveness is forage choice behavior, age of first foraging, and conditioned learning response. Pheromones and hormones are significant components in social insect systems associated with the regulation of colony-level and individual foraging behavior. Bees were treated to different exposure regimes of queen and brood pheromones and their sucrose responsiveness measured. Bees reared with queen or brood pheromone were less responsive than controls. Our results suggest responsiveness to sucrose is a physiologically, neuronally mediated response. Orally administered octopamine significantly reduced sucrose response thresholds. Change in response to octopamine was on a time scale of minutes. The greatest separation between octopamine treated and control bees occurred 30 min after feeding. There was no significant sucrose response difference to doses ranging from 0.2 g to 20 g of octopamine. Topically applied methoprene significantly increased sucrose responsiveness. Handling method significantly affected sucrose responsiveness. Bees that were anesthetized by chilling or CO₂ treatment were significantly more responsive than control bees 30 min after handling. Sixty minutes after handling there were no significant treatment differences. We concluded that putative stress effects of handling were blocked by anesthetic.Abbreviations BP brood pheromone - JH juvenile hormone - OA octopamine - PER proboscis extension response - PER-RT proboscis extension response threshold - QMP queen mandibular pheromone     

Olfactory interference during inhibitory backward pairing in honey bees

Background

Restrained worker honey bees are a valuable model for studying the behavioral and neural bases of olfactory plasticity. The proboscis extension response (PER; the proboscis is the mouthpart of honey bees) is released in response to sucrose stimulation. If sucrose stimulation is preceded one or a few times by an odor (forward pairing), the bee will form a memory for this association, and subsequent presentations of the odor alone are sufficient to elicit the PER. However, backward pairing between the two stimuli (sucrose, then odor) has not been studied to any great extent in bees, although the vertebrate literature indicates that it elicits a form of inhibitory plasticity.

Methodology/Principal Findings

If hungry bees are fed with sucrose, they will release a long lasting PER; however, this PER can be interrupted if an odor is presented 15 seconds (but not 7 or 30 seconds) after the sucrose (backward pairing). We refer to this previously unreported process as olfactory interference. Bees receiving this 15 second backward pairing show reduced performance after a subsequent single forward pairing (excitatory conditioning) trial. Analysis of the results supported a relationship between olfactory interference and a form of backward pairing-induced inhibitory learning/memory. Injecting the drug cimetidine into the deutocerebrum impaired olfactory interference.

Conclusions/Significance

Olfactory interference depends on the associative link between odor and PER, rather than between odor and sucrose. Furthermore, pairing an odor with sucrose can lead either to association of this odor to PER or to the inhibition of PER by this odor. Olfactory interference may provide insight into processes that gate how excitatory and inhibitory memories for odor-PER associations are formed.     

Imidacloprid-induced facilitation of the proboscis extension reflex habituation in the honeybee.

Imidacloprid is a new insecticide from the family of the neonicotinoids, which interact with the insect nicotinic acetylcholine receptor. The effects of imidacloprid at a dose that does not affect sensory or motor functions are studied on non-associative learning abilities in the honeybee. The behavioral procedure is the habituation of the proboscis extension reflex (PER). Imidacloprid topically applied on the thorax (1 microl) at the doses of 5, 10, and 20 ng/bee induces an increase of the gustatory threshold defined as the lowest concentration of a sucrose solution applied to the antennae able to elicit the PER. The ability of the honeybee to move in an open-field-like apparatus is impaired at the doses of 2.5, 5, 10, and 20 ng/bee. These effects are amplified with time and reach a maximum 60 min after application. The lowest dose of 1.25 ng/bee has no effect on the gustatory function but increases the motor activity and facilitates the PER habituation independently of time. This result fits our hypothesis that a slight activation of the cholinergic system with a low dose of imidacloprid can facilitate a simple form of learning in the honeybee.     

草甘膦与微塑料对西方蜜蜂生存和糖反应行为的影响

为分析草甘膦与微塑料对西方蜜蜂Apis mellifera存活和行为的影响。本研究利用50%蔗糖溶液配置的400 mg/L草甘膦、50 mg/L微塑料以及两者混合物,分别饲喂1日龄的工蜂;每日记录西方蜜蜂的存活率,20 d后通过伸吻反应测定它们对蜜蜂嗅觉学习行为的影响。结果发现400 mg/L草甘膦处理蜜蜂20 d后,与对照组相比,蜜蜂存活率下降了22%,伸吻比率下降了30%;50 mg/L微塑料处理,与对照组相比,蜜蜂存活率下降了9%,伸吻比率下降了10%;用400 mg/L草甘膦与50 mg/L微塑料协同处理20 d后,与对照组相比,蜜蜂死亡率为50%、伸吻比率下降了45%左右。结果表明草甘膦和微塑料均会导致西方蜜蜂存活率降低,影响其糖反应行为,而两者协同处理对西方蜜蜂的影响更显著。     

Effects of age and blood sugar levels on the proboscis extension of the blow fly Phormia regina

In some insects the proboscis is extended to imbibe a sugar solution if the concentration of sugar applied to the chemosensilla exceeds the behavioural threshold value. Recently, I found a reversal of the threshold values of this "proboscis extension reflex" (PER) in the blow fly (Phormia regina M.) for glucose and fructose. It depended on maturation and physiological conditions, both of which are explicable in terms of changing concentration of haemolymph trehalose. The direct injection of trehalose into the fly haemocoele brought about a dramatic shift of the threshold values of PER measured on tarsi or labellar sensilla, suggesting a strong dependence of PER on the blood sugar level. Using the tip-recording method, the dose-response (impulse frequency) curves for glucose and fructose were obtained on individual largest labellar chemosensilla. The curves for glucose and fructose crossed at one point because the former had a steeper gradient and higher maximum response than the latter. Injection experiments with trehalose were also carried out to test for changes in gustatory response. The shifting of the behavioural dose-response curves for glucose and fructose two hours after injection of 1 M trehalose (2 μl) into the haemocoele of the fly was associated with significant reduction in responsiveness of labellar chemosensilla to glucose, but less so to fructose. No change in responsiveness was found following injection of mannose. A hypothesis to explain the reversal relation of the PER thresholds, based on a shift in the firing rate in gustatory sensilla and possibly also interneurons, is discussed.     

Effects of cold immobilization and recovery period on honeybee learning, memory, and responsiveness to sucrose

In addition to human error and variation in laboratory conditions, there are numerous factors that can complicate comparisons among studies. Furthermore, differences in how experimental methods are executed can make it difficult to distinguish between effects of focal versus extraneous variables. Insect neural function is commonly evaluated using Pavlovian conditioning techniques; learning and memory in many species can be assessed using the proboscis extension reflex (PER). However, there are significant inconsistencies in methods used to immobilize insects prior to PER tests. We compared responses of honeybees immobilized in a refrigerator, on ice, and in a freezer, and evaluated influence of recovery interval before testing. Ice-chilling weakly decreased learning (response to an originally neutral odor) more so than refrigeration or freezing, but not 24-h recall of odor. We found no significant differences in responsiveness to sucrose relative to cooling method, but responsiveness was significantly lower among honeybees left to recover for only 0.75 h versus 1.5 or 3 h. Finally, we observed increased responsiveness to sucrose and geraniol between June and August. Our results suggest that inconsistencies in cold immobilization methods could confound interpretation and comparison of results from a large body of work on honeybee learning and memory.     

亚致死剂量杀虫剂对意蜂工蜂嗅觉敏感性的影响

喙伸反应(PER)试验适用于评价杀虫剂对蜜蜂行为的影响。本实验利用喙伸反应研究了亚致死剂量(LD50/100～LD50/10)溴氰菊酯和吡虫啉对意蜂Apis mellifera ligustica L.工蜂嗅觉敏感性的影响。结果发现,经口饲喂溴氰菊酯5ng和10ng后,工蜂对0.1%的蔗糖溶液的敏感性显著下降(P<0.05),水应激指数降低,但对0.3%,1%,3%,10%和30%的蔗糖溶液的敏感性没有显著变化;而经口饲喂吡虫啉0.3ng和0.6ng后,工蜂对上述各浓度蔗糖溶液的敏感性变化不明显,但其水应激指数升高。     

Comparative Sucrose Responsiveness in Apis mellifera and A. cerana Foragers

In the European honey bee, Apis mellifera, pollen foragers have a higher sucrose responsiveness than nectar foragers when tested using a proboscis extension response (PER) assay. In addition, Africanized honey bees have a higher sucrose responsiveness than European honey bees. Based on the biology of the Eastern honey bee, A. cerana, we hypothesized that A. cerana should also have a higher responsiveness to sucrose than A. mellifera. To test this hypothesis, we compared the sucrose thresholds of pollen foragers and nectar foragers in both A. cerana and A. mellifera in Fujian Province, China. Pollen foragers were more responsive to sucrose than nectar foragers in both species, consistent with previous studies. However, contrary to our hypothesis, A. mellifera was more responsive than A. cerana. We also demonstrated that this higher sucrose responsiveness in A. mellifera was not due to differences in the colony environment by co-fostering two species of bees in the same mixed-species colonies. Because A. mellifera foragers were more responsive to sucrose, we predicted that their nectar foragers should bring in less concentrated nectar compared to that of A. cerana. However, we found no differences between the two species. We conclude that A. cerana shows a different pattern in sucrose responsiveness from that of Africanized bees. There may be other mechanisms that enable A. cerana to perform well in areas with sparse nectar resources.     

Olfactory conditioning of proboscis activity in Drosophila melanogaster

Olfactory learning and memory processes in Drosophila have been well investigated with aversive conditioning, but appetitive conditioning has rarely been documented. Here, we report for the first time individual olfactory conditioning of proboscis activity in restrained Drosophila melanogaster. The protocol was adapted from those developed for proboscis extension conditioning in the honeybee Apis mellifera. After establishing a scale of small proboscis movements necessary to characterize responses to olfactory stimulation, we applied Pavlovian conditioning, with five trials consisting of paired presentation of a banana odour and a sucrose reward. Drosophila showed conditioned proboscis activity to the odour, with a twofold increase of percentage of responses after the first trial. No change occurred in flies experiencing unpaired presentations of the stimuli, confirming an associative basis for this form of olfactory learning. The adenylyl cyclase mutant rutabaga did not exhibit learning in this paradigm. This protocol generated at least a short-term memory of 15 min, but no significant associative memory was detected at 1 h. We also showed that learning performance was dependent on food motivation, by comparing flies subjected to different starvation regimes.     

Muscle degeneration in the posteclosional development of a Drosophila mutant, abnormal proboscis extension reflex C (aperC)

The aperC (abnormal proboscis extension reflex C) mutation in Drosophila melanogaster causes a defect in the proboscis extension reflex (PER) in aged flies. Young flies of the mutant show an apparently normal PER. When aperCTF36 mutants were reared at 25 degrees C, the flies became unable to extend the proboscis at Day 5 of eclosion, but within a few days, many of them recovered the PER. When reared at 18 degrees C, the mutants showed a defective PER, but did not show the recovery. At 29 degrees C, only a limited number of the mutant flies showed the defective PER. Histological inspection of the mutant revealed that the degeneration occurs in a pair of muscles, the rostral protractors, which are involved in the extension of the rostrum. The degeneration of the muscle was observed in the mutant by polarizing light microscopy. The cross striation disappeared from the central portion of the muscle fibers at Day 3. Birefringence of the fibers also disappeared. At Day 10 the degenerated muscle fibers showed regeneration. The PER was closely correlated with the degree of muscle degeneration and regeneration. Temperature-shift experiments indicated that the temperature-sensitive-period of the aper CTF36 mutation occurs around Days 2-4 after eclosion. Results indicate that the aperC+ gene regulates the posteclosional maintenance of the muscle fibers.     

Proboscis behavioral response of four honey bee Apis species towards different concentrations of sucrose,glucose, and fructose

Honey bees forage for pollen and nectar. Sugar is an important stimulus for foraging and a major source of energy for honey bees. Any differential response of bees to different concentrations of sugary nectar can affect their foraging. The sugar responsiveness of Apis species (Apis dorsata, Apis florea, and Apis cerana) was determined in comparison to that of Apis mellifera by evaluating the proboscis extension response (PER) with eight serial concentrations (0.00001, 0.0001, 0.001, 0.01, 0.1, 0.5, 1.0, and 1.5 M) of sucrose, glucose and fructose. Nectar foragers of bee species (A. dorsata, A. florea, A. cerana, and A. mellifera) exhibited an equal response for sucrose, glucose, and fructose, with no significant differences in their PER at all tested concentrations of these sugars within the same species. The inter-species comparison between Apis species revealed the differential responsiveness to the different concentrations of sugars, and the lowest concentration at which a response occurs was considered as the response threshold of these bee species for sugar solutions. A. mellifera presented significantly higher responsiveness than A. dorsata to low concentrations (0.00001, 0.0001, 0.001, 0.01, and 0.1 M) of sucrose, glucose and fructose. A. mellifera displayed a significantly higher response to water than A. dorsata. A. florea and A. mellifera presented no significant difference in their responsiveness to sucrose, glucose, and fructose at all tested concentrations, and their water responsiveness was also significantly at par but relatively higher in A. mellifera than in A. florea. Likewise, the responsiveness of A. cerana and A. mellifera to different concentrations of sucrose, glucose and fructose was significantly at par with no difference in their water responsiveness. This study represents preliminary research comparing the response of different honey bee species to three sugar types at different concentrations. The results imply that the native species are all better adapted than A. mellifera under local climate conditions.     

Central control of tarsal thresholds for proboscis extension in the blowfly

ABSTRACT. The literature contains contrary reports with respect to the control of the proboscis extension of blowflies in response to stimulatory solutions. Control could be effected centrally, peripherally or by a combination of both. In this report a comparison of (i) behavioural responsiveness to sucrose (measured by tarsal thresholds for proboscis extension) and (ii) sensitivity of tarsal sugar receptors (measured from 0.1 to 1.1 s after stimulus onset) both before and 1 h after feeding of male Protophormia terraenovae suggests that the observed decrease in behavioural responsiveness after feeding is due to central inhibition rather than to a decline in peripheral sensitivity.     

Associative visual learning, color discrimination, and chromatic adaptation in the harnessed honeybee Apis mellifera L.

We studied associative visual learning in harnessed honeybees trained with monochromatic lights associated with a reward of sucrose solution delivered to the antennae and proboscis, to elicit the proboscis extension reflex (PER). We demonstrated five properties of visual learning under these conditions. First, antennae deprivation significantly increased visual acquisition, suggesting that sensory input from the antennae interferes with visual learning. Second, covering the compound eyes with silver paste significantly decreased visual acquisition, while covering the ocelli did not. Third, there was no significant difference in the visual acquisition between nurse bees, guard bees, and foragers. Fourth, bees conditioned with a 540-nm light stimulus exhibited light-induced PER with a 618-nm, but not with a 439-nm light stimulus. Finally, bees conditioned with a 540-nm light stimulus exhibited PER immediately after the 439-nm light was turned off, suggesting that the bees reacted to an afterimage induced by prior adaptation to the 439-nm light that might be similar to the 540-nm light.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

The effect of genotype on response thresholds to sucrose and foraging behavior of honey bees (Apis mellifera L.)

Honey bee foragers were tested for their proboscis extension response (PER) to water and varying solutions of sucrose. Returning pollen and nectar foragers were collected at the entrance of a colony and were assayed in the laboratory. Pollen foragers had a significantly higher probability of responding to water and to lower concentrations of sucrose. Bees derived from artificially selected high- and low-pollen-hoarding strains were also tested using the proboscis extension assay. Returning foragers were captured and tested for PERs to 30% sucrose. Results demonstrated a genotypic effect on PERs of returning foragers. The PERs of departing high- and low-strain foragers were consistent with those of returning foragers. The PERs were related to nectar and water reward perception of foragers. High strain bees were more likely to return with loads of water and lower concentrations of sucrose than foragers from the low pollen strain. Low-strain bees were more likely to return empty. We identified a previously mapped genomic region that contains a variable quantitative trait locus that appears to influence sucrose response thresholds. These studies demonstrate a gene-brain-behavior pathway that can be altered as a consequence of colony-level selection for quantities of stored food. Accepted: 3 September 1997     

Circadian rhythm of proboscis extension responsiveness in the blowfly: central control of threshold changes

ABSTRACT. The proboscis extension response of the blowfly, Protophormia terraenovae (R.-D.), elicited by stimulation of the tarsi with sucrose, was studied behaviourally under LD 12:12. A diel rhythm in responsiveness was found that occurred at successively higher daily mean levels as the test flies became increasingly starved. This rhythm persisted for at least one cycle in constant light, demonstrating it to be truly endogenous and circadian. The tarsal chemo-receptors concerned in initiating the response were examined electrophysiologically in whole fly preparations. No daily change occurred in the response of these receptors to sucrose. Hence central control of the behavioural rhythm of response to sucrose is inferred, and a model is proposed which incorporates a circadian oscillator into the currently accepted model for the control of proboscis extension thresholds.     

Effects of substituted phenylethylamines on blowfly feeding behavior

The pharmacology of adult Phormia regina (Meigen) feeding behavior was explored by injecting candidate drugs into starved blowflies and then determining their responsiveness to aqueous sucrose, via the proboscis extension reflex. d-Amphetamine caused responsiveness to fall dramatically, while related drugs and biogenic amines had varying effects. When d-amphetamine-treated flies were fed 1 M sucrose, they consumed significantly more than control flies. Electrophysiological studies demonstrated that the responses of tarsal sugar receptor neurons to aqueous sucrose were not significantly altered by d-amphetamine. These observations are compatible with our hypothesis that octopamine positively modulates blowfly feeding behavior and suggest that other aromatic biogenic amines affect feeding behavior in this insect.