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.     

Ingestion of sugar diets correlates with spike activity in labellar chemosensilla of the flesh-fly, Neobellieria (= Sarcophaga ) bullata

Abstract .Female 2-day-old Neobellieria (= Sarcophaga ) bullata (Parker) (Diptera: Sarcophagidae) were exposed to different concentrations of sucrose, glucose and fructose in a single-choice potometer, and the volume ingested in the first hour was measured. Nerve spike activity in response to the same sugars was recorded from medium labellar taste hairs of similar flies by tip-recording. Two classes of chemosensory cells responded to sucrose, glucose and fructose. Cell 1 showed an increasing spike activity with sugar concentration, whereas cell 2 did not; cell 1 was identified as the 'sugar cell'.
For both spike activity in cell 1 and feeding, sucrose was the most stimulatory sugar. The dose–response curves for glucose and fructose crossed over at about 200 m m . At higher concentrations, glucose was more stimulatory for both cell 1 and for feeding, and at lower concentrations, fructose. The pattern of spike activity supports a separate location on the sensory cells of receptors for pyranose and fructose forms of sugar. The strong correlation between volume ingested and spike activity indicates that sugar feeding is controlled by sensory input from the 'sugar' cells of labellar chemosensilla. Moreover, the results suggest that the flies do not distinguish between these sugars except by apparent 'sweetness'.     

Two Gr genes underlie sugar reception in Drosophila

We have analyzed the molecular basis of sugar reception in Drosophila. We define the response spectrum, concentration dependence, and temporal dynamics of sugar-sensing neurons. Using in situ hybridization and reporter gene expression, we identify members of the Gr5a-related taste receptor subfamily that are coexpressed in sugar neurons. Neurons expressing reporters of different Gr5a-related genes send overlapping but distinct projections to the brain and thoracic ganglia. Genetic analysis of receptor genes shows that Gr5a is required for response to one subset of sugars and Gr64a for response to a complementary subset. A Gr5a;Gr64a double mutant shows no physiological or behavioral responses to any tested sugar. The simplest interpretation of our results is that Gr5a and Gr64a are each capable of functioning independently of each other within individual sugar neurons and that they are the primary receptors used in the labellum to detect sugars.     

A specific receptor site for glycerol, a new sweet tastant for Drosophila: structure-taste relationship of glycerol in the labellar sugar receptor cell

Glycerol, a linear triol, is a sweet tastant for mammals but it has not previously been recognized to stimulate the sense of taste in insects. Here we show by electrophysiological experimentation that it effectively stimulates the labellar sugar receptor cell of Drosophila. We also show that in accord with the electrophysiological observations, the behavioral feeding response to glycerol is dose dependent. 3-Amino-1,2-propanediol inhibited the response of the sugar receptor cell to glycerol, specifically and competitively, while it had almost no effect on responses to sucrose, D-glucose, D-fructose and trehalose. In the null Drosophila mutant for the trehalose receptor (DeltaEP19), the response to glycerol showed no change, in sharp contrast with a characteristic drastic decrease in the response to trehalose. The glycerol concentration-response curves for I-type and L-type labellar hairs were statistically indistinguishable, while those for sucrose, D-glucose, D-fructose and trehalose were clearly different. These all indicate the presence of a specific receptor site for glycerol. The glycerol site was characterized by comparing the effectiveness of various derivatives of glycerol. Based on this structure-taste relationship of glycerol, a model is proposed for the glycerol site including three subsites and two steric barriers, which cannot accommodate carbon-ring containing sugars such as D-glucose.     

Insoluble alpha-glucosidase: possible pyranose site of the sugar receptor of the labellar of the blowfly, Phormia regina.

The insoluble α-glucosidase activity was examined in three kinds of preparations of the fly labella: labellar integuments with intact chemosensilla, fragments of nerve bundles, and sensilla cut off from the labella.α-Glucosidases having a similar affinity for substrates to isozyme P-II were found in each preparation.Kinetics, inhibition by tris(hydroxymethyl)amino methane (Tris), and pH dependence were examined on the activity of both the membrane-fixed and solublized glucosidase isozymes.Most of the insoluble activity was observed in the nerve bundle fractions, which showed the same properties as P-II in the soluble fraction but were not solubilized by chaotropic anion or by addition of EDTA (insoluble P-II). A different type of insoluble α-glucosidase has been suggested to exist in the labellar chemosensilla in addition to insoluble P-II. It showed a very broad pH dependence and had a larger inhibition constant for Tris. It was inferred to be identical with the enzyme found at the tip of the intact chemosensilla. The hypothesis that the above isozyme might be the pyranose site of the sugar receptor was supported.     

A genetic tool kit for cellular and behavioral analyses of insect sugar receptors

Arthropods employ a large family of up to 100 putative taste or gustatory receptors (Grs) for the recognition of a wide range of non-volatile chemicals. In Drosophila melanogaster, a small subfamily of 8 Gr genes is thought to mediate the detection of sugars, the fly's major nutritional source. However, the specific roles for most sugar Gr genes are not known. Here, we report the generation of a series of mutant sugar Gr knock-in alleles and several composite sugar Gr mutant strains, including a sugar blind strain, which will facilitate the characterization of this gene family. Using Ca²⁺ imaging experiments, we show that most gustatory receptor neurons (GRNs) of sugar blind flies (lacking all 8 sugar Gr genes) fail to respond to any sugar tested. Moreover, expression of single sugar Gr genes in most sweet GRNs of sugar-blind flies does not restore sugar responses. However, when pair-wise combinations of sugar Gr genes are introduced to sweet GRNs, responses to select sugars are restored. We also examined the cellular phenotype of flies homozygous mutant for Gr64a, a Gr gene previously reported to be a major contributor for the detection of many sugars. In contrast to these claims, we find that sweet GRNs of Gr64a homozygous mutant flies show normal responses to most sugars, and only modestly reduced responses to maltose and maltotriose. Thus, the precisely engineered genetic mutations of single Gr genes and construction of a sugar-blind strain provide powerful analytical tools for examining the roles of Drosophila and other insect sugar Gr genes in sweet taste.     

A genetic tool kit for cellular and behavioral analyses of insect sugar receptors

Arthropods employ a large family of up to 100 putative taste or gustatory receptors (Grs) for the recognition of a wide range of non-volatile chemicals. In Drosophila melanogaster, a small subfamily of 8 Gr genes is thought to mediate the detection of sugars, the fly''s major nutritional source. However, the specific roles for most sugar Gr genes are not known. Here, we report the generation of a series of mutant sugar Gr knock-in alleles and several composite sugar Gr mutant strains, including a sugar blind strain, which will facilitate the characterization of this gene family. Using Ca²⁺ imaging experiments, we show that most gustatory receptor neurons (GRNs) of sugar blind flies (lacking all 8 sugar Gr genes) fail to respond to any sugar tested. Moreover, expression of single sugar Gr genes in most sweet GRNs of sugar-blind flies does not restore sugar responses. However, when pair-wise combinations of sugar Gr genes are introduced to sweet GRNs, responses to select sugars are restored. We also examined the cellular phenotype of flies homozygous mutant for Gr64a, a Gr gene previously reported to be a major contributor for the detection of many sugars. In contrast to these claims, we find that sweet GRNs of Gr64a homozygous mutant flies show normal responses to most sugars, and only modestly reduced responses to maltose and maltotriose. Thus, the precisely engineered genetic mutations of single Gr genes and construction of a sugar-blind strain provide powerful analytical tools for examining the roles of Drosophila and other insect sugar Gr genes in sweet taste.     

Two antagonistic gustatory receptor neurons responding to sweet-salty and bitter taste in Drosophila

In Drosophila, gustatory receptor neurons (GRNs) occur within hair-like structures called sensilla. Most taste sensilla house four GRNs, which have been named according to their preferred sensitivity to basic stimuli: water (W cell), sugars (S cell), salt at low concentration (L1 cell), and salt at high concentration (L2 cell). Labellar taste sensilla are classified into three types, l-, s-, and i-type, according to their length and location. Of these, l- and s-type labellar sensilla possess these four cells, but most i-type sensilla house only two GRNs. In i-type sensilla, we demonstrate here that the first GRN responds to sugar and to low concentrations of salt (10-50 mM NaCl). The second GRN detects a range of bitter compounds, among which strychnine is the most potent; and also to salt at high concentrations (over 400 mM NaCl). Neither type of GRN responds to water. The detection of feeding stimulants in i-type sensilla appears to be performed by one GRN with the combined properties of S+L1 cells, while the other GRN detects feeding inhibitors in a similar manner to bitter-sensitive L2 cells on the legs. These sensilla thus house two GRNs having an antagonistic effect on behavior, suggesting that the expression of taste receptors is segregated across them accordingly.     

Two antagonistic gustatory receptor neurons responding to sweet‐salty and bitter taste in Drosophila

In Drosophila, gustatory receptor neurons (GRNs) occur within hair‐like structures called sensilla. Most taste sensilla house four GRNs, which have been named according to their preferred sensitivity to basic stimuli: water (W cell), sugars (S cell), salt at low concentration (L1 cell), and salt at high concentration (L2 cell). Labellar taste sensilla are classified into three types, l‐, s‐, and i‐type, according to their length and location. Of these, l‐ and s‐type labellar sensilla possess these four cells, but most i‐type sensilla house only two GRNs. In i‐type sensilla, we demonstrate here that the first GRN responds to sugar and to low concentrations of salt (10–50 mM NaCl). The second GRN detects a range of bitter compounds, among which strychnine is the most potent; and also to salt at high concentrations (over 400 mM NaCl). Neither type of GRN responds to water. The detection of feeding stimulants in i‐type sensilla appears to be performed by one GRN with the combined properties of S + L1 cells, while the other GRN detects feeding inhibitors in a similar manner to bitter‐sensitive L2 cells on the legs. These sensilla thus house two GRNs having an antagonistic effect on behavior, suggesting that the expression of taste receptors is segregated across them accordingly. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2004     

Sugar receptors in Drosophila

The detection and discrimination of chemical compounds in potential foods are essential sensory processes when animals feed. The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of mostly nonvolatile chemicals that include sugars, a diverse group of toxic compounds present in many inedible plants and spoiled foods, and pheromones [1-6]. With the exception of a trehalose (GR5a) and a caffeine (GR66a) receptor [7-9], the functions of GRs involved in feeding are unknown. Here, we show that the Gr64 genes encode receptors for numerous sugars. We generated a fly strain that contained a deletion for all six Gr64 genes (DeltaGr64) and showed that these flies exhibit no or a significantly diminished proboscis extension reflex (PER) response when stimulated with glucose, maltose, sucrose, and several other sugars. The only considerable response was detected when Gr64 mutant flies were stimulated with fructose. Interestingly, response to trehalose is also abolished in these flies, even though they contain a functional Gr5a gene, which has been previously shown to encode a receptor for this sugar [8, 9]. This observation indicates that two or more Gr genes are necessary for trehalose detection, suggesting that GRs function as multimeric receptor complexes. Finally, we present evidence that some members of the Gr64 gene family are transcribed as a polycistronic mRNA, providing a mechanism for the coexpression of multiple sugar receptors in the same taste neurons.     

Peripheral and central interactions between sugar,water, and salt receptors of the blowfly, Phormia regina

The peripheral and central nervous interactions between the sugar, water, and salt receptors of the blowfly were investigated electrophysiologically by simultaneously recording from the labellar chemoreceptors and the extensor muscle of the haustellum. Simultaneous stimulation of two water receptors with 10 mM LiCl resulted in a motor response even though stimulating the same two sensilla separately with 10 mM LiCl did not. There was a linear decrease in the motor response to two sensilla stimulation as the salt concentration in the stimulating solution increased. Although stimulating two sensilla simultaneously with 200 mM NaCl gave no motor response, simultaneously stimulating two sensilla with 10 mM LiCl and a third with 200 mM NaCl gave a greater response than did two sensilla stimulation with 10 mM LiCl alone, indicating cross-channel summation between the water and salt receptors. Similarly, simultaneously stimulating one sensillum with 100 mM sucrose and another with 10 mM LiCl or 500 mM NaCl gave a larger response than did 100 mM sucrose stimulation alone. The cross-channel summation between the sugar and water receptors was not affected by feeding the flies water. A central excitatory state (CES) which previously had been demonstrated behaviourally was investigated. A stimulation of one sensillum with 10 mM LiCl which previously had been ineffective gave a motor response if proceeded by a stimulation with 1 M sucrose on another sensillum. The effect of the time interval between the sugar and water stimuli was tested, but for intervals of 100 msec to 4 sec no definite correlation was found. In addition, CES with respect to the sugar receptor was demonstrated. The motor response to stimulation of a single sensillum with 100 mM sucrose was enhanced by preceding it with 1 M sucrose stimulation of another sensillum. The motor response to stimulation of two water receptors with 10 mM LiCl was partially inhibited by simultaneously stimulating a third sensillum with 4 M NaCl. Inhibition was also seen when a single sensillum was stimulated with a mixture of 10 mM LiCl and 10 mM sucrose and an adjacent sensillum was simultaneously stimulated with 1 M NaCl. Behavioural experiments showing inhibition of CES by salt were confirmed. Interposing a salt stimulus of 4 M NaCl between the 1 M sucrose and 10 mM LiCl stimuli reduced but did not totally eliminate the motor response to 10 mM LiCl. The basis for peripheral control of the relative activities of the water and salt receptors is discussed. A model is proposed to account for all the receptor interactions in the central nervous system.     

The chemosensitivity of labellar sugar receptor in female Phormia regina is paralleled with ovary maturation: Effects of serotonin

Oogenesis in most adult insects is a nutrient-dependent process involving ingestion of both proteins and carbohydrates that ultimately depends on peripheral input from chemoreceptors.The main goal of this study was to characterize, in the female blowfly Phormia regina, the responsive changes of the labellar chemoreceptors to carbohydrates and proteins in relation to four different stages along the ovarian cycle: (1) immature ovaries, (2) mid-mature ovaries, (3) mature ovaries and ready for egg-laying and (4) post egg-laying ovaries. Then, the possible effects exerted by exogenous serotonin on the chemoreceptor sensitivity profiles were investigated.Our results show that ovary length, width and contraction rate progressively increase from stage 1 to 3, when all these parameters reach their maximum values, before declining in the next stage 4.The sensitivity of the labellar “sugar” chemoreceptors to both sucrose and proteins varies during the ovarian maturation stages, reaching a minimum for sucrose in stage 3, while that to proteins begins. Exogenous 5-HT supply specifically increases the chemoreceptor sensitivity to sugar at the stages 3 and 4, while it does not affect that to proteins.In conclusion, our results provide evidence that in female blowflies the cyclic variations in the sensitivity of the labellar chemosensilla to sugars and proteins are time-related to ovarian development and that during the stages 3 and 4 the responsiveness of the sugar cell to sucrose is under serotonergic control.     

Taste perception and coding in Drosophila

BACKGROUND: Discrimination between edible and contaminated foods is crucial for the survival of animals. In Drosophila, a family of gustatory receptors (GRs) expressed in taste neurons is thought to mediate the recognition of sugars and bitter compounds, thereby controlling feeding behavior. RESULTS: We have characterized in detail the expression of eight Gr genes in the labial palps, the fly's main taste organ. These genes fall into two distinct groups: seven of them, including Gr66a, are expressed in 22 or fewer taste neurons in each labial palp. Additional experiments show that many of these genes are coexpressed in partially overlapping sets of neurons. In contrast, Gr5a, which encodes a receptor for trehalose, is expressed in a distinct and larger set of taste neurons associated with most chemosensory sensilla, including taste pegs. Mapping the axonal targets of cells expressing Gr66a and Gr5a reveals distinct projection patterns for these two groups of neurons in the brain. Moreover, tetanus toxin-mediated inactivation of Gr66a- or Gr5a-expressing cells shows that these two sets of neurons mediate distinct taste modalities-the perception of bitter (caffeine) and sweet (trehalose) taste, respectively. CONCLUSION: Discrimination between two taste modalities-sweet and bitter-requires specific sets of gustatory receptor neurons that express different Gr genes. Unlike the Drosophila olfactory system, where each neuron expresses a single olfactory receptor gene, taste neurons can express multiple receptors and do so in a complex Gr gene code that is unique for small sets of neurons.     

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.     

Food selection in adults and larvae of three species of Lepidoptera: a behavioural and electrophysiological study

Adults and larvae of Spodoptera littoralis, Heliothis virescens and Heliothis armigera were tested with a range of sugars, amino acids, sugar alcohols and allelochemics. Feeding behaviour was correlated with the electrophysiological responses of maxillary styloconic sensilla in the larvae and proboscis styloconic sensilla in the adult. The neural response was more vigorous in larvae than in adults but otherwise the spectra of responses were similar in the two life stages. Phagostimulants and antifeedants stimulated maximally different sensilla in larvae but not in adults. The responses of adult sensilla to sugars and amino acids were significantly correlated to behaviour in all three species, but only in H. armigera was there a similar correlation with the sugar alcohols and allelochemics.     

Biochemical and physiological evidence that calmodulin is involved in the taste response of the sugar receptor cells of the blowfly, Phormia regina

The gustatory system is essential for almost all animals. However, the signal transduction mechanisms have not yet been fully elucidated. We isolated labellar chemosensilla from blowfly, Phormia regina, and purified calcium binding proteins from the water soluble fraction. The most abundant calcium-binding protein was calmodulin. To investigate the role of calmodulin in taste transduction, electrophysiological responses were recorded with the calmodulin inhibitor, W-7. When we stimulated the labellar chemosensillum with sucrose plus W-7, a dose-dependent decrease of impulse frequency was observed when the concentration was <50 microM. In addition, when W-7 at 50 microM or higher concentration was added, an initial short-term impulse generation from the sugar receptor cell was observed, but this was followed by a silent period. When the sensillum was stimulated with W-7 plus a membrane-permeable cGMP analog, dibtyryl-cGMP or 8-bromo-cGMP, impulses of the sugar receptor cell were induced but the frequency was decreased. By the sidewall-recording method, we observed that the receptor potential induced by sucrose stimulation was decreased by W-7 in the sugar receptor cell, and corresponded with a disappearance of impulses. These data strongly suggest that the cGMP-gated channel generating receptor potential in the sugar receptor cell requires calmodulin for its gating.     

Electrophysiological response of honey bee labial palp contact chemoreceptors to sugars and electrolytes

ABSTRACT. The sensilla chaetica on segments II, III and IV of honey-bee labial palps were investigated electrophysiologically. The responses (spikes/s) correlated with the log of the concentrations of sucrose, glucose, fructose, NaCl, KCl and LiCl, but not with CaCl₂ or MgCl₂, which gave inconsistent responses. The firing rates were higher and thresholds lower to the sugars than to the electrolytes. The sensitivity of the segments fell in the order: III > II > IV for most of the stimulants, which elicited responses in the order: sucrose > glucose = fructose' KCl > LiCl > NaCl. The sensilla adapted logarithmically with time. No synergism of response was noted when mixed-sugar solutions were applied, but inhibition of response was seen when glucose–sucrose, fructose–sucrose, and glucose–fructose–sucrose mixtures were applied. None of the sensilla tested responded to water.     

The interaction of external and internal receptors on the feeding behaviour of the blowfly, Phormia regina

The interaction of sensory activity from internal gut stretch receptors and from external labellar chemosensory hairs has been studied both behaviourally and electrophysiologically in the control of proboscis extension of the blowfly, Phormia regina. Labellar thresholds for proboscis extension, tested behaviourally, do not change significantly up to an hour after feeding in contrast to tarsal thresholds which rise quickly after feeding. Motor activity of the extensor muscle of the haustellum was recorded simultaneously with sensory activity from labellar sensilla. The mean number of muscle spikes per response and the sensory input necessary to trigger a response do not vary with starvation, feeding, or sectioning of the recurrent nerve. Activity of internal stretch receptors seem to interact with tarsal sensory input but apparently do not modulate motor responses triggered by labellar sensory input.     

Electrophysiological characterization of responses from gustatory receptor neurons of sensilla chaetica in the moth Heliothis virescens

Discrimination of edible and noxious food is crucial for survival in all organisms. We have studied the physiology of the gustatory receptor neurons (GRNs) in contact chemosensilla (insect gustatory organs) located on the antennae of the moth Heliothis virescens, emphasizing putative phagostimulants and deterrents. Sucrose and the 2 bitter substances quinine and sinigrin elicited responses in a larger proportion of GRNs than inositol, KCl, NaCl, and ethanol, and the firing thresholds were lowest for sucrose and quinine. Variations in GRN composition in individual sensilla occurred without any specific patterns to indicate specific sensillum types. Separate neurons showed excitatory responses to sucrose and the 2 bitter substances quinine and sinigrin, implying that the moth might be able to discriminate bitter substances in addition to separating phagostimulants and deterrents. Besides being detected by separate receptors on the moth antennae, the bitter tastants were shown to have an inhibitory effect on phagostimulatory GRNs. Sucrose was highly appetitive in behavioral studies of proboscis extension, whereas quinine had a nonappetitive effect in the moths.     

Protein-sensitive elements of the labellar sensillas of Musca domestica flies

Using electrophysiological technique of registration of impulse activity in chemoreceptive cells of the labellar sensillae of the housefly, it has been demonstrated that taste hairs are not uniform in their properties. They differ from each other by the set of receptive elements which exhibit different sensitivity, range of selectivity and pattern of impulse activity. It was shown that albumen solution (10(-5) M) evokes the activity in 1--2 cells of a sensilla which are classified as water and sugar receptors. Among these receptors, protein-sensitive and protein-insensitive cells may be distinguished. Considering the inhomogeneity of sugar receptor sites, it was suggested that chemo receptive membranes in most sensitive to protein cells contain more numerous fructose receptive sites, that glucose ones.