A single pair of pharyngeal neurons functions as a commander to reject high salt in Drosophila melanogaster.

Salt (NaCl), is an essential nutrient for survival, while excessive salt can be detrimental. In the fruit fly, Drosophila melanogaster, internal taste organs in the pharynx are critical gatekeepers impacting the decision to accept or reject a food. Currently, our understanding of the mechanism through which pharyngeal gustatory receptor neurons (GRNs) sense high salt are rudimentary. Here, we found that a member of the ionotropic receptor family, Ir60b, is expressed exclusively in a pair of GRNs activated by high salt. Using a two-way choice assay (DrosoX) to measure ingestion volume, we demonstrate that IR60b and two co-receptors IR25a and IR76b are required to prevent high salt consumption. Mutants lacking external taste organs but retaining the internal taste organs in the pharynx exhibit much higher salt avoidance than flies with all taste organs but missing the three IRs. Our findings highlight the vital role for IRs in a pharyngeal GRN to control ingestion of high salt.

A single pair of pharyngeal neurons functions as a commander to reject high salt in Drosophila melanogaster.

Salt is an essential nutrient for survival, while excessive NaCl can be detrimental. In the fruit fly, Drosophila melanogaster, internal taste organs in the pharynx are critical gatekeepers impacting the decision to accept or reject a food. Currently, our understanding of the mechanism through which pharyngeal gustatory receptor neurons (GRNs) sense high salt are rudimentary. Here, we found that a member of the ionotropic receptor family, Ir60b, is expressed exclusively in a pair of GRNs activated by high salt. Using a two-way choice assay (DrosoX) to measure ingestion volume, we demonstrate that IR60b and two coreceptors IR25a and IR76b, are required to prevent high salt consumption. Mutants lacking external taste organs but retaining the internal taste organs in the pharynx exhibit much higher salt avoidance than flies with all taste organs but missing the three IRs. Our findings highlight the vital role for IRs in a pharyngeal GRN to control ingestion of high salt.

Protocol for binary food choice assays using Drosophila melanogaster.

Food preference is a fundamental behavior for animals to choose nutritious foods while rejecting foods containing toxins. Here, we describe binary food choice assays using Drosophila melanogaster, which are straightforward approaches for the characterization of two-way choice tastants. We detail the preparation of flies and dye-containing food, followed by the binary-choice feeding assays and the determination of the preference index (PI). This protocol is simple, sensitive, and reproducible in qualitatively detecting attractive or aversive characteristics toward any two-way choice tastants. For complete details on the use and execution of this protocol, please refer to Aryal et al. (2022).

Drosophila TRPγ is required in neuroendocrine cells for post-ingestive food selection.

The mechanism through which the brain senses the metabolic state, enabling an animal to regulate food consumption, and discriminate between nutritional and non-nutritional foods is a fundamental question. Flies choose the sweeter non-nutritive sugar, L-glucose, over the nutritive D-glucose if they are not starved. However, under starvation conditions, they switch their preference to D-glucose, and this occurs independent of peripheral taste neurons. Here, we found that eliminating the TRPγ channel impairs the ability of starved flies to choose D-glucose. This food selection depends on trpγ expression in neurosecretory cells in the brain that express diuretic hormone 44 (DH44). Loss of trpγ increases feeding, alters the physiology of the crop, which is the fly stomach equivalent, and decreases intracellular sugars and glycogen levels. Moreover, survival of starved trpγ flies is reduced. Expression of trpγ in DH44 neurons reverses these deficits. These results highlight roles for TRPγ in coordinating feeding with the metabolic state through expression in DH44 neuroendocrine cells.

Molecular and neuronal mechanisms for amino acid taste perception in the Drosophila labellum.

Amino acids are essential nutrients that act as building blocks for protein synthesis. Recent studies in Drosophila have demonstrated that glycine, phenylalanine, and threonine elicit attraction, whereas tryptophan elicits aversion at ecologically relevant concentrations. Here, we demonstrated that eight amino acids, including arginine, glycine, alanine, serine, phenylalanine, threonine, cysteine, and proline, differentially stimulate feeding behavior by activating sweet-sensing gustatory receptor neurons (GRNs) in L-type and S-type sensilla. In turn, this process is mediated by three GRs (GR5a, GR61a, and GR64f), as well as two broadly required ionotropic receptors (IRs), IR25a and IR76b. However, GR5a, GR61a, and GR64f are only required for sensing amino acids in the sweet-sensing GRNs of L-type sensilla. This suggests that amino acid sensing in different type sensilla occurs through dual mechanisms. Furthermore, our findings indicated that ecologically relevant high concentrations of arginine, lysine, proline, valine, tryptophan, isoleucine, and leucine elicit aversive responses via bitter-sensing GRNs, which are mediated by three IRs (IR25a, IR51b, and IR76b). More importantly, our results demonstrate that arginine, lysine, and proline induce biphasic responses in a concentration-dependent manner. Therefore, amino acid detection in Drosophila occurs through two classes of receptors that activate two sets of sensory neurons in physiologically distinct pathways, which ultimately mediates attraction or aversion behaviors.

Ionotropic receptors mediate nitrogenous waste avoidance in Drosophila melanogaster.

Ammonia and its amine-containing derivatives are widely found in natural decomposition byproducts. Here, we conducted biased chemoreceptor screening to investigate the mechanisms by which different concentrations of ammonium salt, urea, and putrescine in rotten fruits affect feeding and oviposition behavior. We identified three ionotropic receptors, including the two broadly required IR25a and IR76b receptors, as well as the narrowly tuned IR51b receptor. These three IRs were fundamental in eliciting avoidance against nitrogenous waste products, which is mediated by bitter-sensing gustatory receptor neurons (GRNs). The aversion of nitrogenous wastes was evaluated by the cellular requirement by expressing Kir2.1 and behavioral recoveries of the mutants in bitter-sensing GRNs. Furthermore, by conducting electrophysiology assays, we confirmed that ammonia compounds are aversive in taste as they directly activated bitter-sensing GRNs. Therefore, our findings provide insights into the ecological roles of IRs as a means to detect and avoid toxic nitrogenous waste products in nature.

Cucurbitacin B Suppresses Hyperglycemia Associated with a High Sugar Diet and Promotes Sleep in Drosophila melanogaster.

Secondary metabolites enable plants to protect themselves from herbivorous insects. Among these, cucurbitacin B (cuc-B) is a bitter-tasting compound with promising pharmacological potential. Dietary exposure to cuc-B lowered the hemolymph glucose levels of Drosophila melanogaster fed with a high carbohydrate diet, which is homologous to high blood glucose in humans, and its effect was comparable to that of metformin, a well-known glucose-lowering drug. Furthermore, cuc-B reduced tissue sugar levels and glycogen levels, as well as triacylglycerol levels. Our results thus highlight the potential applicability of this compound to treat chronic metabolic diseases such as diabetes and obesity. Additionally, we analyzed sleep quality and taste-associative memory enhancement after cuc-B and metformin treatment. Both supplements increased nighttime bout length and metformin increased memory consolidation. Therefore, discarded shell of Cucurbitaceae could be processed into health supplements.

Cucurbitacin B Activates Bitter-Sensing Gustatory Receptor Neurons via Gustatory Receptor 33a in Drosophila melanogaster.

The Gustatory system enables animals to detect toxic bitter chemicals, which is critical for insects to survive food induced toxicity. Cucurbitacin is widely present in plants such as cucumber and gourds that acts as an anti-herbivore chemical and an insecticide. Cucurbitacin has a harmful effect on insect larvae as well. Although various beneficial effects of cucurbitacin such as alleviating hyperglycemia have also been documented, it is not clear what kinds of molecular sensors are required to detect cucurbitacin in nature. Cucurbitacin B, a major bitter component of bitter melon, was applied to induce action potentials from sensilla of a mouth part of the fly, labellum. Here we identify that only Gr33a is required for activating bitter-sensing gustatory receptor neurons by cucurbitacin B among available 26 Grs, 23 Irs, 11 Trp mutants, and 26 Gr-RNAi lines. We further investigated the difference between control and Gr33a mutant by analyzing binary food choice assay. We also measured toxic effect of Cucurbitacin B over 0.01 mM range. Our findings uncover the molecular sensor of cucurbitacin B in Drosophila melanogaster. We propose that the discarded shell of Cucurbitaceae can be developed to make a new insecticide.

Transient Receptor Potential Channels and Metabolism.

Transient receptor potential (TRP) channels are nonselective cationic channels, conserved among flies to humans. Most TRP channels have well known functions in chemosensation, thermosensation, and mechanosensation. In addition to being sensing environmental changes, many TRP channels are also internal sensors that help maintain homeostasis. Recent improvements to analytical methods for genomics and metabolomics allow us to investigate these channels in both mutant animals and humans. In this review, we discuss three aspects of TRP channels, which are their role in metabolism, their functional characteristics, and their role in metabolic syndrome. First, we introduce each TRP channel superfamily and their particular roles in metabolism. Second, we provide evidence for which metabolites TRP channels affect, such as lipids or glucose. Third, we discuss correlations between TRP channels and obesity, diabetes, and mucolipidosis. The cellular metabolism of TRP channels gives us possible therapeutic approaches for an effective prophylaxis of metabolic syndromes.