Odor information transfer in the stingless bee<Emphasis Type="Italic"> Melipona quadrifasciata</Emphasis>: effect of in-hive experiences on classical conditioning of proboscis extension

A recent study showed that the stingless bee Melipona quadrifasciata could learn to discriminate odors in a classical conditioning of proboscis extension response (PER). Here we used this protocol to investigate the ability of these bees to use olfactory information obtained within the colony in an experimental context: the PER paradigm. We compared their success in solving a classical differential conditioning depending on the previous olfactory experiences received inside the nest. We found that M. quadrifasciata bees are capable of transferring the food-odor information acquired in the colony to a differential conditioning in the PER paradigm. Bees attained higher discrimination levels when they had previously encountered the rewarded odor associated to food inside the hive. The increase in the discrimination levels, however, was in some cases unspecific to the odor used indicating a certain degree of generalization. The influence of the food scent offered at a field feeder 24 h before the classical conditioning could also be seen in the discrimination attained by the foragers in the PER setup, detecting the presence of long-term memory. Moreover, the improved performance of recruited bees in the PER paradigm suggests the occurrence of social learning of nectar scents inside the stingless bees’ hives.     

Olfactory conditioning with single chemicals in the German Cockroach, Blattella germanica (Dictyoptera: Blattellidae)

Many insects find resources by means of the olfactory cues of general odors after learning. To evaluate behavioral responses to the odor of a particular chemical after learning with reward or punishment quantitatively, we developed a standardized odor-training method in the German cockroach, Blattella germanica (Linnaeus), an important urban pest species. A classical olfactory conditioning procedure for a preference test was modified to become applicable to a single odor, by which a (?)-menthol or vanillin odor was independently associated with sucrose (reward) or sodium chloride solution (punishment). The strength of the association with the odor was evaluated with the increase or decrease in visit frequencies to the odor source after olfactory conditioning. The frequency increased after (?)-menthol was presented with a reward, while it did not change with the rewarded vanillin odor. With both odors, the frequency decreased significantly after training with a punishment. These results indicate that cockroaches learn a single compound odor presented as a conditioned stimulus, although the association of the odor with a reward or punishment depends on the chemical. This olfactory conditioning method can not only facilitate the analysis of cockroach behavior elicited by a learned single chemical odor, but also quantify the potential attractiveness or repellency of the chemical after learning.     

Testing for Odor Discrimination and Habituation in Mice

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the mouse can discriminate between non-social odors and social odors, whether the mouse habituates to a repeatedly presented odor, and whether the mouse demonstrates dishabituation when presented with a novel odor. Since many social behavior tests measure the experimental animal’s response to a familiar or novel mouse, false positives can be avoided by establishing that the animals can detect and discriminate between social odors. There are similar considerations in learning tests such as fear conditioning that use odor to create a novel environment or olfactory cues as an associative stimulus. Deficits in the olfactory system would impair the ability to distinguish between contexts and to form an association with an olfactory cue during fear conditioning. In the odor habitation/dishabituation test, the mouse is repeatedly presented with several odors. Each odor is presented three times for two minutes. The investigator records the sniffing time directed towards the odor as the measurement of olfactory responsiveness. A typical mouse shows a decrease in response to the odor over repeated presentations (habituation). The experimenter then presents a novel odor that elicits increased sniffing towards the new odor (dishabituation). After repeated presentation of the novel odor the animal again shows habituation. This protocol involves the presentation of water, two or more non-social odors, and two social odors. In addition to reducing experimental confounds, this test can provide information on the function of the olfactory systems of new knockout, knock-in, and conditional knockout mouse lines.     

Olfactory responses ofTrichogramma maidis Pint,et Voeg.: Effects of chemical cues and behavioral plasticity

Oriented responses of Trichogramma maidisPint, et Voeg. to airborne odors were observed in a four-armed olfactometer. Experiments were carried out with odors of host eggs, the sex pheromone of Ostrinia nubilalisHbn, and maize extract, offered singly or in combination, both to naive wasps and to wasps previously exposed to the tested odor during an oviposition experience. The exploratory behavior in the olfactometer was quantified by means of a computer program which performed a space-time analysis of the insect 's movements. Whereas the naive wasps did not respond to the odor of the eggs, the synthetic sex pheromone of O. nubilalis,or the maize extract presented singly, they did react to a mixture of these three volatile cues. Prior oviposition in the odor of maize extract or in the combination of odors induced an increased preference toward the conditioning scent. This phenomenon did not occur when the wasps were conditioned to egg odor or sex pheromone alone. These results suggest that females can learn to associate some olfactory cues with the presence of the host. Immediately following the presentation of the combination of odors, a strong attraction of experienced wasps occurred; it decreased as the experiment progressed and finally reached the level presented by naive insects. Adult conditioning to the combination of odors also resulted in reduced variability in the behavioral responses.     

Altered representation of the spatial code for odors after olfactory classical conditioning; memory trace formation by synaptic recruitment

In the olfactory bulb of vertebrates or the homologous antennal lobe of insects, odor quality is represented by stereotyped patterns of neuronal activity that are reproducible within and between individuals. Using optical imaging to monitor synaptic activity in the Drosophila antennal lobe, we show here that classical conditioning rapidly alters the neural code representing the learned odor by recruiting new synapses into that code. Pairing of an odor-conditioned stimulus with an electric shock-unconditioned stimulus causes new projection neuron synapses to respond to the odor along with those normally activated prior to conditioning. Different odors recruit different groups of projection neurons into the spatial code. The change in odor representation after conditioning appears to be intrinsic to projection neurons. The rapid recruitment by conditioning of new synapses into the representation of sensory information may be a general mechanism underlying many forms of short-term memory.     

Olfactory discrimination of eight food flavors in the capuchin monkey (Cebus apella): Comparison between fruity and fishy odors

Operant conditioning was used to investigate how primates discriminate between odor qualities. Eight artificial food flavors, selected from either a “Fishy” or “Aroma/Fruity” category, were used. During the presence of one of the two odors S+ or S−, the monkey was reinforced by pushing a response key when S + was presented. The tufted capuchins discriminated most accurately when both odors were Aroma. Discrimination was more accurate when S+ was Fruity odor and S− was Fishy odor. When both odors were Fishy, discrimination could not be acquired in 20 sessions. All of the flavors used, except apple, were novel for the subjects, which suggests that capuchins can innately discriminate among them. The data also suggest that Aroma odors are more salient than Fishy odors. The results also suggested an innate aversion to Fishy odors.     

Classical olfactory conditioning in the cockroach Periplaneta americana

We established a classical conditioning procedure for the cockroach, Periplaneta americana, by which odors were associated with reward or punishment. Cockroaches underwent differential conditioning trials in which peppermint odor was associated with sucrose solution and vanilla odor was associated with saline solution. Odor preference of cockroaches was tested by allowing them to choose between peppermint and vanilla sources. Cockroaches that had undergone one set of differential conditioning trials exhibited a significantly greater preference for peppermint odor than did untrained cockroaches. Memory formed by three sets of differential conditioning trials, with an inter-trial interval of 5 min, was retained at least 4 days after conditioning. This conditioning procedure was effective even for cockroaches that had been harnessed in plastic tubes. This study shows, for the first time in hemimetaborous insects, that both freely moving and harnessed insects are capable of forming olfactory memory by classical conditioning procedure. This procedure may be useful for future electrophysiological and pharmacological studies aimed at elucidation of neural mechanisms underlying olfactory learning and memory.     

Conditioned sexual arousal in a nonhuman primate

Conditioning of sexual arousal has been demonstrated in several species from fish to humans but has not been demonstrated in nonhuman primates. Controversy exists over whether nonhuman primates produce pheromones that arouse sexual behavior. Although common marmosets copulate throughout the ovarian cycle and during pregnancy, males exhibit behavioral signs of arousal, demonstrate increased neural activation of anterior hypothalamus and medial preoptic area, and have an increase in serum testosterone after exposure to odors of novel ovulating females suggestive of a sexually arousing pheromone. Males also have increased androgens prior to their mate's ovulation. However, males presented with odors of ovulating females demonstrate activation of many other brain areas associated with motivation, memory, and decision making. In this study, we demonstrate that male marmosets can be conditioned to a novel, arbitrary odor (lemon) with observation of erections, and increased exploration of the location where they previously experienced a receptive female, and increased scratching in post-conditioning test without a female present. This conditioned response was demonstrated up to a week after the end of conditioning trials, a much longer lasting effect of conditioning than reported in studies of other species. These results further suggest that odors of ovulating females are not pheromones, strictly speaking and that marmoset males may learn specific characteristics of odors of females providing a possible basis for mate identification.     

Body odor similarity in noncohabiting twins

There is currently considerable interest in biometric approaches using human odor as a marker of disease or genetic individuality. Body odor is also thought to be used during mate choice to select genetically compatible mates. The idea that body odor reveals information about both genetic identity and genetic similarity is most readily tested by examining odor in twin pairs. However, although this idea can be traced back 130 years to Francis Galton in 1875, most studies using dogs fail to control for shared environmental effects associated with cohabitation. Here we show that odors of identical twins (but not dizygotic twins) can be matched by human sniffers at rates better than chance, even when the twins are living apart. In addition, matching frequencies for identical twin odors were not significantly different from those for duplicate odors from the same individual. These results indicate an important genetic influence on body odor and the potential for developing technologies for human odor printing in relation to underlying genotype.     

Generalization mediates sensitivity to complex odor features in the honeybee

Animals use odors as signals for mate, kin, and food recognition, a strategy which appears ubiquitous and successful despite the high intrinsic variability of naturally-occurring odor quantities. Stimulus generalization, or the ability to decide that two objects, though readily distinguishable, are similar enough to afford the same consequence, could help animals adjust to variation in odor signals without losing sensitivity to key inter-stimulus differences. The present study was designed to investigate whether an animal's ability to generalize learned associations to novel odors can be influenced by the nature of the associated outcome. We use a classical conditioning paradigm for studying olfactory learning in honeybees to show that honeybees conditioned on either a fixed- or variable-proportion binary odor mixture generalize learned responses to novel proportions of the same mixture even when inter-odor differences are substantial. We also show that the resulting olfactory generalization gradients depend critically on both the nature of the stimulus-reward paradigm and the intrinsic variability of the conditioned stimulus. The reward dependency we observe must be cognitive rather than perceptual in nature, and we argue that outcome-dependent generalization is necessary for maintaining sensitivity to inter-odor differences in complex olfactory scenes.     

Alteration of mouse urinary odor by ingestion of the xenobiotic monoterpene citronellal

Body odors provide a rich source of sensory information for other animals. There is considerable evidence to suggest that short-term fluctuations in body odor can be caused by diet; however, few, if any, previous studies have demonstrated that specific compounds can directly mask or alter mouse urinary odor when ingested and thus alter another animal's behavior. To investigate whether the ingestion of citronellal, a monoterpene aldehyde that produces an intense aroma detected by both humans and mice, can alter mouse urinary odor, mice (C57BL6J) were trained in a Y maze to discriminate between the urinary odors of male donor mice that had ingested either citronellal in aqueous solution or a control solution. Trained mice could discriminate between urinary odors from the citronellal ingestion and control groups. A series of generalization tests revealed that citronellal ingestion directly altered mouse urinary odor. Moreover, trained mice that had successfully discriminated between urinary odors from donor mice of different ages failed to detect age-related changes in urine from male mice that had ingested 50 ppm of citronellal. This study is the first to show that ingestion of a xenobiotic can alter mouse urinary odor and confuse the behavioral responses of trained mice to age-related scents.     

Smell-induced gamma oscillations in human olfactory cortex are required for accurate perception of odor identity

Studies of neuronal oscillations have contributed substantial insight into the mechanisms of visual, auditory, and somatosensory perception. However, progress in such research in the human olfactory system has lagged behind. As a result, the electrophysiological properties of the human olfactory system are poorly understood, and, in particular, whether stimulus-driven high-frequency oscillations play a role in odor processing is unknown. Here, we used direct intracranial recordings from human piriform cortex during an odor identification task to show that 3 key oscillatory rhythms are an integral part of the human olfactory cortical response to smell: Odor induces theta, beta, and gamma rhythms in human piriform cortex. We further show that these rhythms have distinct relationships with perceptual behavior. Odor-elicited gamma oscillations occur only during trials in which the odor is accurately perceived, and features of gamma oscillations predict odor identification accuracy, suggesting that they are critical for odor identity perception in humans. We also found that the amplitude of high-frequency oscillations is organized by the phase of low-frequency signals shortly following sniff onset, only when odor is present. Our findings reinforce previous work on theta oscillations, suggest that gamma oscillations in human piriform cortex are important for perception of odor identity, and constitute a robust identification of the characteristic electrophysiological response to smell in the human brain. Future work will determine whether the distinct oscillations we identified reflect distinct perceptual features of odor stimuli.

Intracranial recordings from human olfactory cortex reveal a characteristic spectrotemporal response to odors, including theta, beta and gamma oscillations, and show that high-frequency responses are critical for accurate perception of odors.     

The role of learning in adult food location by the larval parasitoid,Microplitis croceipes (Hymenoptera: Braconidae)

Wind tunnel experiments were conducted to determine roles of odor learning in food foraging of the larval parasitoid,Microplitis croceipes (Hymenoptera: Braconidae). Females that had neither fed on sucrose water nor experienced any odor and females that had experienced an odor without feeding failed to respond to any odors in a wind tunnel. Most of the females that had fed without an odor also did not respond to odors. However, most of the females that had experienced an odor during feeding on sucrose water flew to the odor. These results indicate that when females experience an odor during feeding, they learn to associate the odor with food and subsequently respond to the odor. As age of females increased, their response to an experienced odor increased, peaked 2 to 5 days after emergence, and then decreased. With an increasing number of odor experiences while feeding, accuracy of females choosing the experienced odor increased. Females that experienced an odor while feeding three to five times chose the experienced odor 90% of the time. When females experienced an odor while feeding five times, the memory of food associated odor lasted at least 2 days. When they experienced food with two odors successively, they could memorize both odors, and multiple experiences did not cause memory interference. Even when females had learned a food-associated odor, their response to the learned odor ceased after several visits on patches containing the odor but no food. Such negative experience may cause switching of food searching to new odors by females.     

Olfactory Associative Learning of the Pupal Parasitoid Pimpla luctuosa Smith (Hymenoptera: Ichneumonidae)

Laboratory experiments were conducted to determine the role of learning in olfactory host searching by the ichneumonid pupal parasitoid, Pimpla luctuosa Smith. Females learned to associate novel odors such as vanilla and strawberry with hosts when they oviposited in at least several hosts with the odors. Repeated experiences of hosts with an odor increased the response of the experienced odor, and females that had experienced host odor seven times responded to the experienced odors 90% of the time. Although the response by females to a learned odor gradually decreased with increasing host-deprivation time, 60% of the females that had experienced host odor 7 days earlier still responded to the experienced odor. Females also learned two separate odors associated with hosts at a time and responded to both odors without a preference for one odor over the other. When trained two separate odors with hosts, females learned the second odor more quickly than the first odor. After females experienced several stings in simulated hosts with the previously learned odor, they ceased to respond to the learned odor, suggesting that repeated unrewarding experiences cause females to cease to respond to the learned odors.     

Odors: implicit memory and performance effects.

In order to assess the influence of odors on human performance and implicit memory for odors, 108 subjects completed a variety of tests in weakly scented (jasmine, lavender or odorless) rooms without having been made aware of the odor. After a 30 min interval the subjects were shown slides of different surroundings, including the room they had been in, and were requested to rate how well a set of 12 odors, including a blank, would fit to these surroundings. Half of these contexts contained visual cues related to two of the presented odors (leather and coffee). After the rating of fit the subjects had to rate the odors for pleasantness, were asked to identify the odors with their correct names and to tell where and when they had last smelled these odors. One subject remembered smelling the odor (jasmine) in the room and was discarded from the analysis of the results for the rating of fit. None of the others reported recollection of the experimental odors. The results showed that in general jasmine had a negative and lavender a positive effect on test performance. If an odor-related visual cue was present in the context, the related odor was always rated highest in fit to that context. Furthermore, the subjects working in rooms with an odor subsequently assigned this odor to the visual context of that room to a significantly higher degree than subjects working in rooms with different odors. Since none of the subjects reported that they had smelled the odor in the rooms where performance testing took place, it was concluded that the memory for these odors was implicit. Further analysis showed that such memory was only found in subjects who were unable to supply the right name for the odor. The possible consequences of this latter finding for understanding the relationship between sensory (episodic) and semantic odor memory are discussed.     

The Odor Awareness Scale: a new scale for measuring positive and negative odor awareness

The Odor Awareness Scale (OAS) is a questionnaire designed to assess individual differences in awareness of odors in the environment. The theory that odor awareness can be distinguished in awareness of negative (to be avoided) odors and positive (to be approached) odors was tested using confirmatory factor analysis (CFA) on the 34-item questionnaire after completion by 525 respondents. CFA (after deletion of 2 items) showed good fit of the 2-factor theory, resulting in a positive awareness subscale (11 items, Cronbach's alpha = .77) and a negative awareness subscale (21 items, Cronbach's alpha = .80). Furthermore, reports of sickness from environmental odors were correlated with the negative odor awareness factor, not the positive odor awareness factor. Respondents scoring high on the overall sum score of the OAS showed significantly better olfactory performance on an odor perception test battery than respondents with a low score. These results suggest a causal relation between awareness of potentially negative odors, olfactory performance and experiencing health effects from environmental odor exposure, that warrants further investigation.     

Task Specialization and Odor Effects on Proboscis Extension Conditioning in Bumblebees (Bombus huntii)

Workers in a social insect colony have distinct experiences that may affect their performance in a learning task. In this study using free-foraging and flight-cage bumblebee Bombus huntii colonies, the strength of olfactory proboscis extension conditioning (PEC) was affected by a bee’s task specialization as a nurse or forager and the stimulus odor. Nurses (n?=?26) learned to respond to the odors 1-hexanol and lavender, but foragers (n?=?25) had inhibited conditioning to both odors. More nurses (73 %) than foragers (48 %) had at least one conditioned response (CR), and nurses displayed significantly more CRs than foragers. As expected, a pseudoconditioned control group (n?=?9) showed very few CRs. Among bees that were given a second day of trials, nurses—but not foragers—showed modest improvement. Such strong inhibition of PEC has not been described in honeybee or bumblebee foragers. The stimulus odor also affected conditioning strength in nurses. Lavender, a familiar odor, elicited earlier and more CRs than 1-hexanol. We propose that learning floral odors in the context of foraging may inhibit PEC in bumblebee foragers, whereas exposure to odors in the honey stores may prime subsequent learning in nurses.     

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.     

Punishment prediction by dopaminergic neurons in Drosophila

The temporal pairing of a neutral stimulus with a reinforcer (reward or punishment) can lead to classical conditioning, a simple form of learning in which the animal assigns a value (positive or negative) to the formerly neutral stimulus. Olfactory classical conditioning in Drosophila is a prime model for the analysis of the molecular and neuronal substrate of this type of learning and memory. Neuronal correlates of associative plasticity have been identified in several regions of the insect brain. In particular, the mushroom bodies have been shown to be necessary for aversive olfactory memory formation. However, little is known about which neurons mediate the reinforcing stimulus. Using functional optical imaging, we now show that dopaminergic projections to the mushroom-body lobes are weakly activated by odor stimuli but respond strongly to electric shocks. However, after one of two odors is paired several times with an electric shock, odor-evoked activity is significantly prolonged only for the "punished" odor. Whereas dopaminergic neurons mediate rewarding reinforcement in mammals, our data suggest a role for aversive reinforcement in Drosophila. However, the dopaminergic neurons' capability of mediating and predicting a reinforcing stimulus appears to be conserved between Drosophila and mammals.     

Context-dependent changes in the perception of odor quality

Ambiguous odor compounds, partly citrus-like and partly woodyin odor character, were seen to change in odor quality whenevaluated in the same session as more prototypical odors. Whentested with characteristically citrus odors, the ambiguous compoundsseemed more woody, and when tested with characteristically woodyodors, the ambiguous odorants were higher in citrus character,an example of perceptual contrast. Response frequency biaseswere ruled out as an explanation for this shift by an experimentin which responses other than citrus and woody ratings wereasked of the subjects during the contextual exposure. Simplesensory adaptation was found to be a potential contributor tothe effect, and a sufficient condition to produce similar shiftsin odor quality. However, adaptation was not a necessary conditionto produce the effect. This was seen in reversed pair experimentsin which the contextual odors were presented after the ambiguousstimuli. The contextual shift was robust—it was obtainedwith different ambiguous odors, contextual (conditioning) odors,numbers of contextual odors, orders of presentation of contextualodors relative to ambiguous odors, scale types, and rating tasksduring the presentation of contextual odors.