桔小实蝇雄成虫联系性嗅觉学习

【目的】为了探究桔小实蝇 Bactrocera dorsalis (Hendel)雄成虫的嗅觉学习能力。【方法】本研究采用经典性嗅觉条件反射训练法（classical olfactory conditioning）在室内对固定的羽化后14-17日龄的桔小实蝇雄成虫进行气味与食物的联合学习训练, 即薄荷精油和10%蔗糖溶液联合的奖赏性训练（appetitive conditioning）以及甲基丁香酚(methyl eugenol, ME)和饱和盐溶液联合的惩罚性训练（aversive conditioning）,并以伸喙反射行为（proboscis extension reflex, PER）作为学习与否的判定标准。【结果】经过奖赏性训练后,桔小实蝇雄成虫对薄荷精油的伸喙反射率可从0%增加至68%;而经过惩罚性训练后,桔小实蝇对甲基丁香酚的伸喙反射率可从100%降低至36.54%,且这种伸喙反射率的变化是通过气味条件刺激（conditioned stimulus）和食物非条件刺激（unconditioned stimulus）的对称性联合而产生的。【结论】结果表明,桔小实蝇雄性成虫具有较强的联系性嗅觉学习能力,并且两种刺激的联合是形成学习记忆的必要条件。     

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.     

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.     

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.     

Classical Olfactory Conditioning in the Oriental Fruit Fly,Bactrocera dorsalis

The oriental fruit fly, Bactrocera dorsalis, is a serious pest of fruits and vegetables. Methyl eugenol (ME), a male attractant, is used to against this fly by mass trapping. Control effect may be influenced by learning, which could modify the olfactory response of the fly to this attractant. To collect the behavioral evidence, studies on the capability of this fly for olfactory learning are necessary. We investigated olfactory learning in male flies with a classical olfactory conditioning procedure using restrained individuals under laboratory conditions. The acquisition of the proboscis extension reflex was used as the criterion for conditioning. A high conditioned response level was found in oriental fruit flies when an odor was presented in paired association with a sucrose reward but not when the odor and sucrose were presented unpaired. We also found that the conditioning performance was influenced by the odor concentration, intertrial interval, and starvation time. A slight sensitization elicited by imbibing sucrose was observed. These results indicate that oriental fruit flies have a high capacity to form an olfactory memory as a result of classical conditioning.     

Behavioral studies on tarsal gustation in honeybees: sucrose responsiveness and sucrose-mediated olfactory conditioning

Although the forelegs of honeybees are one of their main gustatory appendages, tarsal gustation in bees has never been systematically studied. To provide a more extensive account on honeybee tarsal gustation, we performed a series of behavioral experiments aimed at characterizing (1) tarsal sucrose sensitivity under different experimental conditions and (2) the capacity of tarsal sucrose stimulation to support olfactory conditioning. We quantified the proboscis extension reflex to tarsal sucrose stimulation and to odors paired with tarsal sucrose stimulation, respectively. Our experiments show that tarsal sucrose sensitivity is lower than antennal sucrose sensitivity and can be increased by starvation time. In contrast, antennae amputation decreases tarsal sucrose sensitivity. Furthermore, we show that tarsal sucrose stimulation can support olfactory learning and memory even if the acquisition level reached is relatively low (40%).     

Effects of acute sublethal exposure to coumaphos or diazinon on acquisition and discrimination of odor stimuli in the honey bee (Hymenoptera: Apidae)

Two organophosphate compounds, coumaphos and diazinon, were examined for effects of sublethal exposure on odor learning and generalization in honey bees, Apis mellifera L. Using proboscis extension response training as a measure of odor learning and discrimination, a series of two experiments tested whether these compounds would inhibit bees from learning a new odor or discriminating between different odors. Bees were exposed to coumaphos or diazinon in acetone applied to the thorax, or to coumaphos or diazinon in hexane injected intracranially. At no dose tested or exposure method used was coumaphos shown to inhibit acquisition of a novel odor stimulus, although it was shown to slightly reduce discriminatory ability when given by intracranial injection. Diazinon had effects on odor learning at several small doses, and a small injected dose was shown to significantly inhibit learning of an odor stimulus paired with a sucrose reward. When bee head acetylcholineasterase activity was measured after dermal applications of both pesticides, only the higher doses of diazinon showed reduced activity, indicating that externally-applied coumaphos shows no significant effect on bee brain acetylcholinesterase activity. These data suggest that acute application of coumaphos has only slight nonlethal effects upon the behavior of honey bees and should have little effect upon bee tasks that involve odor learning.     

Can Honey Bees Learn the Removal of a Stimulus as a Conditioning Cue?

Experiments investigated a Pavlovian conditioning situation where the presence and absence of the stimulus are reversed temporally with respect to the presentation of a reward. Instead of a conditioned stimulus (e.g. odor) signaling the presence of a reward, the stimulus (e.g. odor) is present in the environment except just prior to the presence of the reward. Thus, the absence of the stimulus, or offset of the stimulus (e.g. absence of odor), serves as a conditioned stimulus and is the reward cue. Honey bees (Apis mellifera) were used as a model invertebrate system, and the proboscis‐conditioning paradigm was used as the test procedure. Using both simple Pavlovian conditioning and discrimination‐learning protocols, animals learned to associate the onset of an odor as conditioned stimuli when paired with a sucrose reward. They could also learn to associate the onset of a puff of air with a sucrose reward. However, bees could not associate the offset of an order stimulus with the presentation of a sucrose reward in either a simple conditioning or a discrimination‐learning situation. These results support the model that a very different cognitive architecture is used by invertebrates to deal with certain environmental situations, including signaled avoidance.     

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.     

Olfactory perception in honeybees: Concatenated and mixed odorant stimuli,concentration, and exposure effects

Summary Here we present results obtained from 7 different series of experiments, all employing odor conditioning of proboscis extension in worker honeybees and each designed to address a particular question involving olfactory perception. The questions relate to: temporal complexity of odor cues; effects of concentration, suppression, and/or potentiation in mixture perception; acquisition and extinction rates, as well as levels of generalization associated with aliphatic compounds that have the same functional groups or same alkyl radical length; and the effects of continuous exposure to odorants in the first several days of adult life on various learning and discrimination tasks involving olfactory perception. From the data obtained in these experiments we were able to conclude the following: First, worker honeybees have a limited ability to perceive complex temporal odor-quality patterns in olfactory stimuli — they learn to associate the quality of only the last part of the stimulus with a sucrose reward. Second, we confirm that citral is qualitatively different in several perceptual contexts involving odor learning and conditioning and our results help elucidate the nature of these differences as they relate to learning, discrimination, mixture perception, and continuous exposure to particular odorants. Third, we appear to have uncovered some important perceptual differences between functional groups attached to the first as opposed to the second carbon atom of alkyl radicals. Finally, we failed to uncover any significant effects relating to continuous exposure to odorants during the first several days of a worker's adult life, despite evidence that considerable sensory development takes place during this period. Thus ontogenetic changes to the peripheral system due to environmental effects appear to leave basic perceptual systems unaltered.     

A simple and reliable test of olfactory learning and memory in mice

The present paper describes a quick and efficient method for assessing olfactory discrimination learning in mice. In training mice received trials in which one odor (CS+) was paired with sugar and another odor (CS-) was paired with no sugar. When the mice were subsequently placed in a chamber with CS+ odor at one end and CS- odor at the other, they spent more time digging in CS+ than in CS- odor. In Experiment 2 mice trained with this procedure and tested after 60 days also spent more time digging in CS+ than CS- in the test phase, indicating that this olfactory discrimination task is effective for assessing long-term memory. In addition to the outbred strain of CD1 mice used in Experiments 1 and 2, C57Bl/6NCr/BR and DBA/2NCr/BR mice used in Experiment 3 also acquired this learned odor discrimination. Moreover, Experiment 4 showed that DBA animals were capable of acquiring this odor discrimination after receiving only two training trials (one exposure each to CS+ and CS-) per day for 4 days.     

Pavlov's cockroach: classical conditioning of salivation in an insect

Secretion of saliva to aid swallowing and digestion is an important physiological function found in many vertebrates and invertebrates. Pavlov reported classical conditioning of salivation in dogs a century ago. Conditioning of salivation, however, has been so far reported only in dogs and humans, and its underlying neural mechanisms remain elusive because of the complexity of the mammalian brain. We previously reported that, in cockroaches Periplaneta americana, salivary neurons that control salivation exhibited increased responses to an odor after conditioning trials in which the odor was paired with sucrose solution. However, no direct evidence of conditioning of salivation was obtained. In this study, we investigated the effects of conditioning trials on the level of salivation. Untrained cockroaches exhibited salivary responses to sucrose solution applied to the mouth but not to peppermint or vanilla odor applied to an antenna. After differential conditioning trials in which an odor was paired with sucrose solution and another odor was presented without pairing with sucrose solution, sucrose-associated odor induced an increase in the level of salivation, but the odor presented alone did not. The conditioning effect lasted for one day after conditioning trials. This study demonstrates, for the first time, classical conditioning of salivation in species other than dogs and humans, thereby providing the first evidence of sophisticated neural control of autonomic function in insects. The results provide a useful model system for studying cellular basis of conditioning of salivation in the simpler nervous system of insects.     

Does pollen function as a reward for honeybees in associative learning?

The ability to learn an association between floral characteristics such as its odor, color and shape and a reward such as nectar is key to honeybee foraging success. Here, we tested if also pollen could function as a reward for associative learning in honeybees. We found that large proportions of bees with and without field experience showed an unconditioned response, the extension of the proboscis, after touching their antennae with bee-collected pollen. Furthermore, bees readily learn to associate an odor with pollen in a classical conditioning assay. We suggest that pollen might play an important role as a reward for free-flying bees. Received 12 February 2008; revised 17 June 2008; accepted 15 July 2008. C. Grüter, A. Arenas: Both authors contributed equally to this work.     

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.     

The influence of combinations of encoded amino acids on associative learning in the honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis> L.

This study addresses the influence of combinations of two encoded amino acids with opposite, memory-inhibiting and memory-enhancing, effects on short-term and long-term memory formation in the honeybee. Experimentation was based on the classical proboscis extension response conditioning by a single-trial exposure to clove odor with a sucrose solution reward. The presence of the acquired conditioned response was tested 1 min (short-term memory) and 180 min (long-term memory) after the learning trial. The data obtained suggest a stimulatory (memory-enhancing) effect of the combination of two amino acids, individual effects of which are opposite. The stimulatory amino acid was present in the mixture in all the trials (8 combinations) at a subthreshold concentration, i.e. it did not influence memory formation. In some trials, the stimulatory effect of an amino acid mixture (for example, stimulatory aspartic acid combined with inhibitory lysine or serine) significantly exceeded that of a single stimulatory amino acid applied at a threshold concentration. Interestingly, the effect of amino acid combinations on memory formation in honeybees resembles their effect on cell proliferation in rat tissue explants of various origin.     

Effects of Proteinase Inhibitor Ingestion on Survival,Learning Abilities and Digestive Proteinases of the Honeybee

The impact on beneficial insects of proteinase inhibitors expressed in pest-resistant transgenic crops needs to be assessed before the release of these plants into the environment. Three proteinase inhibitors, suitable for incorporation into oilseed rape, were tested on worker bees: the chicken egg white cystatin, oryzacystatin I (OCI) and Bowman-Birk soyabean inhibitor (BBI). Ingestion of low doses of the inhibitors did not cause short-term mortality, and a conditioned proboscis extension assay showed that olfactory learning performances were unchanged when the inhibitors were added to the reward. Long-term ingestion of BBI or OCI did not disrupt total digestive proteolytic activity, but ingestion of BBI induced a new proteinase form, suggesting the existence of a mechanism of control of proteinase synthesis in the honeybee.     

Volatile exposure within the honeybee hive and its effect on olfactory discrimination

Honeybees of different ages and reproductive castes cohabit in the hive where they are exposed to many odors that might affect associative learning. Our aim was to analyze the role of odors pre-exposed as volatiles on appetitive learning in honeybees of different ages and search for their long-term effect both under natural and laboratory conditions. By evaluating memory acquisition and retention through a differential proboscis extension response conditioning, we found that hive-exposed odors offered as a reinforced conditioned stimulus during training promoted a learning-reduced effect [latent inhibition (LI)]. On the other hand, no effect was found when the non-reinforced conditioned stimulus was pre-exposed. The LI effect varied with the odor identity. However, only slight differences were found with the age of the bees. Exposure-conditioning intervals longer than 24 h did not show an LI effect unless the odor concentration was increased or exposure was prolonged. Our results show that pre-exposed volatiles could either reduce learning performance, if this odor is later associated with food, or be irrelevant in the case that alternative scented resources circulate within the colony. The differential effects found according to the olfactory exposure characteristics could strongly influence the propagation of chemosensory information within the hive.     

Different functions of two alarm substances in the honeybee

In the honeybee, isopentyl acetate and 2-heptanone are described as alarm substances. We asked whether both substances have a similar role by testing the effect of their exposure on the appetitive proboscis extension reflex and on the aversive stinging reflex. In the appetitive context of sucrose stimulation no differences were found between isopentyl acetate and 2-heptanone. Small amounts of isopentyl acetate or 2-heptanone (3 microl of 1:9 dilution) yielded a response similar to that of a non-exposed control. Larger amounts of both substances (125 microl of 1:9 dilutions) as well as mixtures led to a decrease of responsiveness to sucrose. In the aversive context of electrical stimulation, significant differences between isopentyl acetate and 2-heptanone were found. Exposure to a small amount of isopentyl acetate (3 microl of 1:9 dilution) or to a large amount of 2-heptanone (125 microl of 1:9 dilution) led to an increase of responsiveness to the electric shock. Larger quantities of isopentyl acetate (125 microl of 1:9 dilution) decreased the responsiveness to the shock. 2-Heptanone never decreased the responsiveness to the shock. Our results indicate that isopentyl acetate and 2-heptanone have different functions even if both are capable of evoking deterrent responses in a defensive context.     

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.     

Conditional suppression of a reflex in Drosophila melanogaster: acquisition and extinction]

In Drosophila it was possible to carry out a negative instrumental conditioning of the tarsal reflex (extension of the proboscis in response to a sucrose stimulation of the forelegs) : the insects could be trained to inhibit this reaction through a repetitive association of its releasing with a consecutive nociceptive stimulation. An experimental extinction procedure allowed the reflex to be resoted.