Effect of an amino acid on feeding preferences and learning behavior in the honey bee, Apis mellifera

Amino acids are common constituents of floral nectars and can be critical components in the diets of insect pollinators. Yet the means through which insects detect amino acids can be complex and arise from pre- and post-ingestive mechanisms. Furthermore, the response to an amino acid can change depending on an insect's nutritional status. Here we use a sensitive feeding assay and Proboscis Extension Response (PER) conditioning in the honey bee to assay the effect of glycine, which is a common constituent of nectars and pollens. Subjects preferred to feed on a sucrose stimulus that contained glycine, and the highest relative preference was recorded for the highest concentration of glycine. However, the highest response rate occurred at lower than maximal concentrations and differed depending on the physiological status of the subjects. These results are consistent with a model in which subjects attempt to maintain a physiological target amount of glycine/amino acid relative to other nutrients. All concentrations of glycine enhanced the rate and magnitude of a conditioned response to an odor in the PER assay, which demonstrates that animals can learn to modify their responses to an odor conditioned stimulus based on the presence of amino acid. This capability would enhance a honey bee's ability to evaluate the quality of floral nectars, which are associated with, among other things, odor cues given off by flowers. In future studies these techniques will allow us to evaluate the physiological roles that amino acids play in honey bee diet and choice behavior.     

Effect of age on the responsiveness of peripheral chemosensory sensilla of the turnip root fly (Delia floralis)

The effect of insect age on the neural responsiveness of gustatory sensilla was investigated. Electrophysiological recordings were obtained from type A and type D sensilla on the pro- and meso-thoracic tarsi, and from sensilla on the labellum of the turnip root fly,Delia floralis (Fallen) in response to potassium chloride, sucrose and sinigrin. The age of the fly did have an effect on the numbers of sensilla responding to the test stimuli and on the magnitude of the response, but there was no consistent pattern in these effects among sensilla. The labellar sensilla were more responsive to sucrose than the tarsal sensilla and the proportion of flies whose labellar sensilla responded to sucrose was initially low, but increased after day 2 of adult life. In contrast, the number of flies whose tarsal sensilla responded to stimulation with sucrose was initially high and decreased as the flies aged. There was a similar decline in the proportion of tarsal sensilla responding to potassium chloride. Neither the proportion of flies whose tarsal sensilla responded to sinigrin nor the magnitude of the response was influenced by the age of the fly. These finding are discussed in relation to the feeding and host selection behaviour of the fly.     

Spatially restricted expression of candidate taste receptors in the Drosophila gustatory system

BACKGROUND: Taste is an important sensory modality in most animals. In Drosophila, taste is perceived by gustatory neurons located in sensilla distributed on several different appendages throughout the body of the animal. Here we show that the gustatory receptors are encoded by a family of at least 54 genes (Gr genes), most of which are expressed exclusively in a small subset of taste sensilla located in narrowly defined regions of the fly's body. RESULTS: BLAST searches with the predicted amino acid sequences of 6 7-transmembrane-receptor genes of unknown function and 20 previously identified, putative gustatory receptor genes led to the identification of a large gene family comprising at least 54 genes. We investigated the expression of eight genes by using a Gal4 reporter gene assay and found that five of them were expressed in the gustatory system of the fly. Four genes were expressed in 1%-4% of taste sensilla, located in well-defined regions of the proboscis, the legs, or both. The fifth gene was expressed in about 20% of taste sensilla in all major gustatory organs, including the taste bristles on the anterior wing margin. Axon-tracing experiments demonstrated that neurons expressing a given Gr gene project their axons to a spatially restricted domain of the subesophageal ganglion in the fly brain. CONCLUSIONS: Our findings suggest that each taste sensillum represents a discrete, functional unit expressing at least one Gr receptor and that most Gr genes are expressed in spatially restricted domains of the gustatory system. These observations imply the potential for high taste discrimination of the Drosophila brain.     

Mechanisms of Carboxylic Acid Attraction in Drosophila melanogaster

Sour is one of the fundamental taste modalities that enable taste perception in animals. Chemoreceptors embedded in taste organs are pivotal to discriminate between different chemicals to ensure survival. Animals generally prefer slightly acidic food and avoid highly acidic alternatives. We recently proposed that all acids are aversive at high concentrations, a response that is mediated by low pH as well as specific anions in Drosophila melanogaster. Particularly, some carboxylic acids such as glycolic acid, citric acid, and lactic acid are highly attractive to Drosophila compared with acetic acid. The present study determined that attractive carboxylic acids were mediated by broadly expressed Ir25a and Ir76b, as demonstrated by a candidate mutant library screen. The mutant deficits were completely recovered via wild-type cDNA expression in sweet-sensing gustatory receptor neurons. Furthermore, sweet gustatory receptors such as Gr5a, Gr61a, and Gr64a-f modulate attractive responses. These genetic defects were confirmed using binary food choice assays as well as electrophysiology in the labellum. Taken together, our findings demonstrate that at least two different kinds of receptors are required to discriminate attractive carboxylic acids from other acids.     

Perception of noxious compounds by contact chemoreceptors of the blowfly,Phormia regina: putative role of an odorant-bindingpProtein

The blowfly, Phormia regina, has sensilla with four contact-chemoreceptor cells and one mechanoreceptor cell on its labellum. Three of the four chemoreceptor cells are called the sugar, the salt and the water receptor cells, respectively. However, the specificity of the remaining chemoreceptor cell, traditionally called the "fifth cell", has not yet been clarified. Referring to behavioral evaluation of the oral toxicity of monoterpenes, we measured the electrophysiological response of the "fifth cell" to these compounds. Of all the monoterpenes examined, D-limonene exhibited the strongest oral toxicity and induced the severest aversive behavior with vomiting and/or excretion in the fly. D-Limonene, when dispersed in an aqueous stimulus solution including dimethyl sulfoxide or an odorant-binding protein (OBP) found in the contact-chemoreceptor sensillum, the chemical sense-related lipophilic ligand-binding protein (CRLBP), evoked impulses from the "fifth cell". Considering the relationship between the aversive effects of monoterpenes and the response of the "fifth cell" to these effects, we propose that the "fifth cell" is a warning cell that has been differentiated as a taste system for detecting and avoiding dangerous foods. Here we suggest that in the insect contact-chemoreceptor sensillum, CRLBP carries lipophilic members of the noxious taste substances to the "fifth cell" through the aqueous sensillum lymph. This insect OBP may functionally be analogous to the von Ebner's grand protein in taste organs of mammals.     

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic,Biomedical, and Agricultural Research

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.     

Gustatory organs of Drosophila melanogaster: fine structure and expression of the putative odorant-binding protein PBPRP2

In Drosophila, as in most insects, gustation is mediated by sensory hairs located on the external and internal parts of the proboscis and on the legs and wings. We describe in detail the organization and ultrastructure of the gustatory sensilla on the labellum and legs and the distribution of PBPRP2, a putative odorant-binding protein, in the gustatory organs of Drosophila. The labellum carries two kinds of sensilla: taste bristles and taste pegs. The former have the typical morphology of gustatory sensilla and can be further subdivided into three morphological subtypes, each with a stereotyped distribution and innervation. Taste pegs have a unique morphology and are innervated by two receptor cells: one mechanoreceptor and the other a putative chemoreceptor cell. PBPRP2 is abundantly expressed in all adult gustatory organs on labellum, legs, and wings and in the internal taste organs on the proboscis. In contrast to olfactory organs, where PBPRP2 is expressed in the epidermis, this protein is absent from the epidermis of labial palps and legs. In the taste bristles of the labellum and legs, PBPRP2 is localized in the crescent-shaped lumen of the sensilla, and not in the lumen where the dendrites of the gustatory neurons are found, making a function in stimulus transport unlikely in these sensilla. In contrast, PBPRP2 in peg sensilla is expressed in the inner sensillum-lymph cavity and is in contact with the dendrites. Thus, PBPRP2 could be involved as a carrier for hydrophobic ligands, e.g., bitter tastants, in these sensilla.     

Behavioural and chemosensory responses of the turnip root fly (Delia floralis) to glucosinolates

This study investigates the behavioural and neural mechanisms involved in the oviposition behaviour of the turnip root fly,Delia floralis (Fallen). Behavioural studies showed that glucosinolates modulated the oviposition behaviour of the flies on artificial leaves as well as the number of eggs laid in the soil at the base of these leaves. Electrophysiological responses to glucosinolates were obtained from type A and type D sensilla on the prothoracic and mesothoracic tarsi, as well as from the long contact sensilla on the labellum. The neural responses from these sensilla were positively correlated with the oviposition behaviour of the flies and with the number of eggs laid. Of the eleven glucosinolates tested in the behavioural and electrophysiological tests, the flies were most responsive to glucobrassicanapin, gluconapin and glucobrassicin. The type D tarsal sensilla were more responsive to the glucosinolates than either the type A tarsal sensilla or the labellar sensilla. The structure-activity investigations showed that slight modifications to the chemical composition of the glucosinolates resulted in changes in neural activity.     

Neurobiology of the fruit fly's circadian clock

Studying the fruit fly Drosophila melanogaster has revealed mechanisms underlying circadian clock function. Rhythmic behavior could be assessed to the function of several clock genes that generate circadian oscillations in certain brain neurons, which finally modulate behavior in a circadian manner. This review outlines how individual circadian pacemaker neurons in the fruit fly's brain control rhythm in locomotor activity and eclosion.     

Dopaminergic modulation of sucrose acceptance behavior in Drosophila

For an animal to survive in a constantly changing environment, its behavior must be shaped by the complex milieu of sensory stimuli it detects, its previous experience, and its internal state. Although taste behaviors in the fly are relatively simple, with sugars eliciting acceptance behavior and bitter compounds avoidance, these behaviors are also plastic and are modified by intrinsic and extrinsic cues, such as hunger and sensory stimuli. Here, we show that dopamine modulates a simple taste behavior, proboscis extension to sucrose. Conditional silencing of dopaminergic neurons reduces proboscis extension probability, and increased activation of dopaminergic neurons increases extension to sucrose, but not to bitter compounds or water. One dopaminergic neuron with extensive branching in the primary taste relay, the subesophageal ganglion, triggers proboscis extension, and its activity is altered by satiety state. These studies demonstrate the marked specificity of dopamine signaling and provide a foundation to examine neural mechanisms of feeding modulation in the fly.     

Adhesion forces measured at the level of a terminal plate of the fly's seta

The attachment pads of fly legs are covered with setae, each ending in small terminal plates coated with secretory fluid. A cluster of these terminal plates contacting a substrate surface generates strong attractive forces that hold the insect on smooth surfaces. Previous research assumed that cohesive forces and molecular adhesion were involved in the fly attachment mechanism. The main elements that contribute to the overall attachment force, however, remained unknown. Multiple local force-volume measurements were performed on individual terminal plates by using atomic force microscopy. It was shown that the geometry of a single terminal plate had a higher border and considerably lower centre. Local adhesion was approximately twice as strong in the centre of the plate as on its border. Adhesion of fly footprints on a glass surface, recorded within 20 min after preparation, was similar to adhesion in the centre of a single attachment pad. Adhesion strongly decreased with decreasing volume of footprint fluid, indicating that the layer of pad secretion covering the terminal plates is crucial for the generation of a strong attractive force. Our data provide the first direct evidence that, in addition to Van der Waals and Coulomb forces, attractive capillary forces, mediated by pad secretion, are a critical factor in the fly's attachment mechanism.     

Optomotor responses of the fly Musca domestica to transient stimuli of edges and stripes

The fly's optomotor response to transient stimuli was studied under open loop conditions. The stimuli used were moving edges and stripes. A comparison of the fly's responses to these stimuli bends to the result that progressive moving patterns (from front to back with respect to the fly) elicit stronger responses than regressive moving ones (from back to front). Edges followed by darkness elicit a stronger response than those followed by light. A narrow, bright or dark stripe and a single edge evoke a similar response, whereas a broad stripe elicitis a stronger response than a single edge.     

How the two eyes add together: monocular properties of the visually guided orientation behaviour of flies

In this account fixation and the torque response to a transient moving stripe of flying femaleMusca domestica with monocular sight was tested. This was made by either covering one eye of the fly with opaque paint or by placing a screen in front of one side of the fly's visual field. A stripe was moved with constant speed once around the fly clockwise and, after a pause, counterclockwise. The torque response of the fly was measured during the motion of the stripe and shortly beforehand. The results demonstrated that the monocular torque response to progressive (from front to back) motion and regressive (from back to front) motion essentially do not differ from the binocular response, except for the region of bionocular overlap. The beginning of the response of a fly with monocular vision to progressive motion is 11 ° (on average) before the direction of flight (0°), which means that the maximal functional binocular overlap of femaleMusca domestica is stretched at least 15° to each side (3.1). In addition, the shape of the monocular torque response to a progressively moving stripe was determined (see Figs. 5Ia and 5IIb). In other experiments similar to the ones described above, a screen was placed on one side of the fly's visual field or then on the other, (instead of covering one eye) and the torque response to the moving stripe was measured. Using this method, a delay response of 90 ms was measured. We suggest that this is the delay of the direction-sensitive component of the torque response, and therefore an additional argument for the existence of two components for the optomotor torque response. Flies with a covered eye or with a screen placed in front of one side of the visual field were able to fixate a single narrow long black stripe. This, however, was possible only when an additional offset signal was added, in order to give the stripe a constant velocity component. As a result there was a shift of the fixation towards the unobscured eye. The shift was small for the monocular flies, and it was larger (13° on average) when the screen was on one side of the fly. A new type of laser torquethrust transducer was developed and is described.     

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     

Testing Drosophila Olfaction with a Y-maze Assay

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.     

A large family of divergent Drosophila odorant-binding proteins expressed in gustatory and olfactory sensilla.

We identified a large family of putative odorant-binding protein (OBP) genes in the genome of Drosophila melanogaster. Some of these genes are present in large clusters in the genome. Most members are expressed in various taste organs, including gustatory sensilla in the labellum, the pharyngeal labral sense organ, dorsal and ventral cibarial organs, as well as taste bristles located on the wings and tarsi. Some of the gustatory OBPs are expressed exclusively in taste organs, but most are expressed in both olfactory and gustatory sensilla. Multiple binding proteins can be coexpressed in the same gustatory sensillum. Cells in the tarsi that express OBPs are required for normal chemosensation mediated through the leg, as ablation of these cells dramatically reduces the sensitivity of the proboscis extension reflex to sucrose. Finally, we show that OBP genes expressed in the pharyngeal taste sensilla are still expressed in the poxneuro genetic background while OBPs expressed in the labellum are not. These findings support a broad role for members of the OBP family in gustation and olfaction and suggest that poxneuro is required for cell fate determination of labellar but not pharyngeal taste organs.     

The course of habituation of the proboscis extension reflex can be predicted by sucrose responsiveness in Drosophila

The proboscis extension reflex (PER) is triggered when insects' gustatory receptors contact appetitive stimuli, so it provides a behavioral readout for perceptual encoding of tastants. Research on the experience dependent modulation of PER in Drosophila has been hindered by the difficulty of obtaining reliable measures of memory-driven change in PER probability in the background of larger changes induced by physiological state. In this study, we showed that the course of PER habituation can be predicted by the degree of sucrose responsiveness in Drosophila. We assessed early response parameters, including the number of proboscis extensions and labellar movements in the first five trials, the trial to start responding, and the trial to make the first stop to quantify responsiveness, which predicted the upcoming pattern of both the short-term and 1 hour memory of PER habituation for individual flies. The cAMP signaling pathway mutant rutabaga displayed deficits in attunement of perceptual salience of sucrose to physiological demands and stimulus-driven sensitization.