Most animals have functionally distinct populations of taste cells, expressing receptors that are tuned to compounds of different valence. This organizational feature allows for discrimination between chemicals associated with specific taste modalities and facilitates differentiating between unadulterated foods and foods contaminated with toxic substances. In the fruit fly D. melanogaster, primary sensory neurons express taste receptors that are tuned to distinct groups of chemicals, thereby activating neural ensembles that elicit either feeding or avoidance behavior. Members of a family of ligand gated receptor channels, the Gustatory receptors (Grs), play a central role in these behaviors. In general, closely related, evolutionarily conserved Gr proteins are co-expressed in the same type of taste neurons, tuned to chemically related compounds, and therefore triggering the same behavioral response. Here, we report that members of the Gr28 subfamily are expressed in largely non-overlapping sets of taste neurons in Drosophila larvae, detect chemicals of different valence and trigger opposing feeding behaviors. We determined the intrinsic properties of Gr28 neurons by expressing the mammalian Vanilloid Receptor (VR1), which is activated by capsaicin, a chemical to which wildtype Drosophila larvae do not respond. When VR1 is expressed in Gr28a neurons, larvae become attracted to capsaicin, consistent with reports showing that Gr28a itself encodes a receptor for nutritious RNA. In contrast, expression of VR1 in two pairs of Gr28b.c neurons triggers avoidance to capsaicin. Moreover, neuronal inactivation experiments show that the Gr28b.c neurons are necessary for avoidance of several bitter compounds. Lastly, behavioral experiments of Gr28 deficient larvae and live Ca2+ imaging studies of Gr28b.c neurons revealed that denatonium benzoate, a synthetic bitter compound that shares structural similarities with natural bitter chemicals, is a ligand for a receptor complex containing a Gr28b.c or Gr28b.a subunit. Thus, the Gr28 proteins, which have been evolutionarily conserved over 260 million years in insects, represent the first taste receptor subfamily in which specific members mediate behavior with opposite valence.
This valuable study focuses on the role of the Gr28 family of insect chemoreceptors. Using the Drosophila larva, the authors show that taste neurons expressing different members of this family of bitter taste receptors trigger opposite behavior – attraction and repulsion. They establish the minimal bitter taste receptor subunit composition needed in these neurons to mediate the repulsion of bitter tastants. The evidence presented is convincing, using well-validated and controlled tools and experiments.
Meaningful animal behavior is established through cooperative engagement of multiple sensory systems. In many insects, the chemosensory system plays a central role in such integration processes. The fruit fly Drosophila melanogaster has served as the primary insect model system for elucidating the molecular basis and neural circuitry of both olfaction and taste, by virtue of the vast genetic resources, amenability to both neurophysiological recording and live imaging, and simple yet powerful behavioral assays, allowing investigators to link genes to chemosensory behavior and neural activity (Montell, 2021).
The Drosophila gustatory system is characterized by several insect specific anatomical and molecular features. For example, taste cells are primary sensory neurons, with dendritic processes that express taste receptors, while long axons project and convey taste information directly to the brain. In adult flies, these neurons, referred to as Gustatory Receptor Neurons (GRNs) are distributed across several appendages, such as labial palps, legs and presumably the antennae. Additionally, some GRNs reside internally, arranged in cell clusters along the pharynx (Amrein, 2016; Joseph and Carlson, 2015; Scott, 2018). Likewise, Drosophila larvae have numerous structures located both on the head surface as well as internally along the larval pharynx, where chemical compounds are assessed during their passage towards the digestive system (Figure 1A)(Apostolopoulou et al., 2015; Kwon et al., 2011; Rist and Thum, 2017).
Fruit flies, like most insects, employ taste receptors encoded by two major gene families, the Gustatory receptor (Gr) and the Ionotropic Receptor (IR) genes, to sense soluble chemicals, such as appetitive food compounds, noxious and toxic chemicals as well as pheromones. Both Gr- and IR- based receptors are thought to form complexes composed of several, and generally different, subunits. Composition of only a few Gr-based taste receptors for sugars and bitter compounds is known (Jiao et al., 2008; Yavuz et al., 2014; Shim et al., 2015; Sung et al., 2017) (Fujii et al., unpublished), and two Gr proteins, Gr21a and Gr63a, are co-expressed in a small subset of olfactory neurons where they form a receptor complex for gaseous carbon dioxide (Jones et al., 2007; Kwon et al., 2007).
The Gr genes represent the largest taste receptor gene family in insects. In D. melanogaster, it is comprised of 60 genes predicted to encode 68 proteins, expression of which has been extensively described (Dahanukar et al., 2007; Dunipace et al., 2001; Fujii et al., 2015; Scott et al., 2001; Weiss et al., 2011). Several Gr genes have been functionally characterized in adult flies using genetic mutations combined with either electrophysiological recordings, Ca2+ imaging studies and behavioral analyses (Amrein, 2016; Montell, 2021), but only a few have been studied in larvae (Apostolopoulou et al., 2016a; Choi et al., 2016, 2020; Mishra et al., 2013, 2018).
The Gr28 gene subfamily is of particular interest for a number of reasons: First, it is one of the most conserved Gr subfamilies, homologs of which can be found across all insect families and even more distant arthropods (Eyun et al., 2017; Fujii et al., 2023; Suzuki et al., 2018). The six Gr28 genes are tightly clustered, with the five Gr28b genes transcribed from distinct promotors and unique exons spliced to shared 2nd and 3rd exons, while Gr28a is a separate transcription unit (Figure 1B). Overall conservation between the Gr28 proteins is high (equal or more than 50% similarity), characteristic for genes generated through recent gene duplication events. Second, the Gr28 genes are expressed not only in the gustatory system of larvae and adult flies, but also in many other organs, especially the central nervous system (CNS) and non-chemosensory neurons of the peripheral nervous system (PNS), suggesting that they have functions beyond gustation and are important to sense chemical signals unrelated to food (Mishra et al., 2018; Thorne and Amrein, 2008). Notably, evidence for such roles has been reported before any direct link to gustatory perception was discovered. Ni and colleagues showed that Gr28b.d is essential for high temperature avoidance in flies (Ni et al., 2013), while Xiang and collaborators found that Gr28 mutant larvae were deficient in UV light avoidance (Xiang et al., 2010). Third, the only known gustatory function for any Gr28 protein thus far is sensing of RNA and ribose by Gr28a, mediated by Gr28a-GAL4 GRNs (Mishra et al., 2018). Remarkably, sensing RNA and ribose is an appetitive taste quality found in other dipteran insects that diverged from Drosophila about 260 million years ago, including flesh flies and mosquitoes, and we showed that Gr28 homologs from A. aegypti and A. gambiae can restore RNA and ribose preference in Drosophila Gr28 mutant larvae when expressed in Gr28a-GAL4 GRNs (Fujii et al., 2023).
Here, we present a detailed expression analysis and functional characterization of the Gr28 genes and the respective GRNs in Drosophila larvae. In addition to Gr28a, three of the five Gr28b genes (Gr28b.a, Gr28b.c and Gr28b.e) are also expressed in the larval taste system. Interestingly, GRNs expressing Gr28a and the three Gr28b genes represent functionally distinct neuronal ensembles, with minimal expression overlap in a single pair of neurons expressing both Gr28a and Gr28b.c. When the mammalian Vanilloid Receptor 1 (VR1) is expressed under the control of specific GAL4 drivers, we found that Gr28a-GAL4 and Gr28b.c-GAL4 neurons mediate opposing taste behavior in the presence of capsaicin, the ligand for VR1. Specifically, Gr28a-GAL4/UAS-VR1 larvae show strong attraction for capsaicin, while Gr28b.c-GAL4/UAS-VR1 larvae show strong avoidance of capsaicin. Neuronal inactivation experiments reveal that the Gr28b.c GRNs are necessary to sense bitter compounds, such as denatonium, quinine, lobeline, and caffeine. Moreover, Ca2+ responses of Gr28b.c GRNs to denatonium and quinine are significantly reduced and avoidance behavior of these two chemicals is diminished in Gr28 mutant larvae, whereas Ca2+ responses and avoidance behavior were not affected when challenged with lobeline and caffeine. This implies that Gr28b proteins are subunits of receptor complexes sensing a subgroup of bitter tasting compounds. In summary, the Gr28 genes encode related Gr proteins mediating both positive and negative valence.
Expression of the Gr28 genes in the larval taste organs
The peripheral chemosensory system of the larvae is subdivided into bilaterally symmetrical, ‘external’ and ‘internal’ taste organs (Stocker, 2008). The three external organs reside near the tip of the head and are organized as paired ganglia, the Dorsal, Terminal and Ventral Organ Ganglia (DOGs, TOGs and VOGs) that house the GRN cell bodies with dendritic extensions in respective sensory organs (DO, TO and VO) at the head surface, while carrying information via their axons to the subesophageal zone (SEZ) in the brain (Figure 1A). The DOG harbors 21 olfactory neurons and 18 presumptive GRNs. For clarity, GRN numbers refer to neurons in one of the two, bilaterally symmetrical taste organs. The GRNs located in the DOG fall into two distinct groups, based on their dendritic extensions: 11 presumptive GRNs, four of which were shown to express Gr genes (Apostolopoulou et al., 2015), send dendrites to the base of the DO, while 7 neurons extend dendrites to the TO (the dorsolateral group, Figure 1A)(Kwon et al., 2011). The TOG contains 30 neurons, with dendrites located in the TO (the distal group). The internal taste structures, referred to as the Dorsal/Ventral Pharyngeal Sense organ (DPS/VPS, 33 neurons) and the Posterior Pharyngeal Sense organs (PPS, 6 neurons) are located along the pharynx and sense chemicals as they are moved towards the digestive system. We note that not all these neurons are confirmed GRNs, either by function or expression of Gr or IR genes, albeit based on their location and anatomy, most are thought to be GRNs (Rist and Thum, 2017; Sánchez-Alcañiz et al., 2018; Stewart et al., 2015).
Our initial expression analysis of the Gr28 genes revealed that four of the six Gr28 genes (Gr28a, Gr28b.a, Gr28b.c and Gr28b.e) were expressed in larval taste organs, in addition to cells in the gut, the brain and non-chemosensory cells of the larvae (Mishra et al., 2018; Thorne and Amrein, 2008). This and all previous Gr expression studies were performed using bimodal expression systems, mostly GAL4/UAS, whereby Gr promotors driving GAL4 are assumed to faithfully reproduce expression of the respective Gr genes. Importantly, we analyzed two or four Gr28-GAL4 insertion lines for each transgene, and at least two generated the same expression pattern (Mishra et al., 2018; Thorne and Amrein, 2008) providing evidence that the drivers reflect a fairly accurate expression profile of respective endogenous genes. To further delineate the putative chemosensory roles of these genes, we performed a more detailed co-expression analysis between the Gr28 genes and the bitter receptor gene Gr66a by combining the GAL4/UAS (labeling Gr28 expressing neurons) with the LexA/lexAop (marking Gr66a neurons) system (Figure 1C and 1D). In the TOG, we found expression of all four Gr28 genes, along with that of Gr66a, which was expressed in 3 or 4 neurons (this number is slightly smaller than the 6 neurons previously reported by (Kwon et al., 2011). GAL4 drivers for Gr28b.a, Gr28b.c and Gr28b.e are co-expressed in a single neuron with Gr66a-LexA (Figure 1C). In contrast, Gr28a-GAL4 is expressed in a distinct TOG neuron than Gr66a-LexA (Figure 1C) and the Gr28b genes, an observation we independently confirmed using a Gr28b.c-LexA driver (Figure 1-figure supplement 1). In the DOG, we find only a single Gr28a-GAL4 neuron, while none of the Gr28b genes, or Gr66a, is expressed there (Figure 1C). None of the Gr28-GAL4 drivers was expressed in the VOG. In the internal sensory organs, Gr66a-LexA is found in one GRN in the DPS/VPS, where it is co-expressed with Gr28b.c-GAL4 as well as Gr28a-GAL4 (Figure 1D). In the PPS, none of the Gr28b genes is expressed, but Gr28a-GAL4 is found in two neurons, one of which also expresses Gr66a-LexA (Figure 1D). In summary, the internal sensory organs can be subdivided in three distinct groups (Figure 1E): Gr66a/Gr28b.c/Gr28a positive neurons, Gr66a/Gr28a positive neurons and Gr28aonly neurons, which contrasts the external sensory organs where Gr28a and the Gr28b genes are expressed in a mutually exclusive fashion.
Subsets of Gr28 neurons mediate opposing feeding behaviors
We previously showed that at least one of the six Gr28 genes is necessary for feeding attraction to RNA, ribonucleosides and ribose using a well-established two choice feeding assay (Mishra et al., 2018)(Figure 2A). Specifically, larvae homozygous mutant for Gr28 (ΔGr28, a deletion of the entire Gr28a gene and more than half of the coding region of all Gr28b genes, Figure 1B), lose their ability to sense these compounds, a phenotype that is restored when single UAS-Gr28 reporter transgenes are expressed in Gr28a-GAL4 neurons. The largely non-overlapping expression of Gr28a and the Gr28b genes suggests that respective neurons represent functionally distinct entities. To investigate this possibility, we took advantage of the mammalian Vanilloid Receptor 1 (VR1), a TRP channel that is activated by capsaicin (Caterina et al., 1997). Drosophila have no VR1 like-gene in their genome and do not respond to capsaicin behaviorally, but flies are attracted to this chemical when a modified VR1 gene (VR1E600K) is expressed in sweet taste neurons (Marella et al., 2006). Thus, we expressed VR1E600K (henceforth referred to UAS-VR1) under the control of the four Gr28-GAL4 drivers (Gr28a-GAL4, Gr28b.a-GAL4, Gr28b.c-GAL4 and Gr28b.e-GAL4) in larvae and tested their response to capsaicin using the two-choice preference assay (Figure 2A). Additionally, we expressed UAS-VR1 in bitter neurons as well as appetitive fructose sensing neurons using respective GAL4 drivers (Gr66a-GAL4 and Gr43aGAL4) (Mishra et al., 2013; Scott et al., 2001), which served as control larvae (note that fructose sensing Gr43aGAL4 neurons are distinct from Gr28a-GAL4 neurons (Mishra et al., 2018)). Just like adult flies, w1118control larvae lacking either a UAS-VR1 reporter, a Gr-GAL4 driver, or both, were unresponsive to 0.1 mM capsaicin, while the positive control larvae expressing UAS-VR1 in either fructose sensing GRNs or bitter taste GRNs showed robust attraction to or avoidance of capsaicin (Figure 2B). Consistent with previous findings, which identified Gr28a GRNs as appetitive neurons (Mishra et al., 2018), larvae expressing UAS-VR1 under the control of Gr28a-GAL4 showed strong appetitive responses to capsaicin (Figure 2B). In contrast, larvae expressing UAS-VR1 under the control of Gr28b.c-GAL4 showed robust avoidance behavior to capsaicin, while expression in the single TOG GRN using either Gr28b.a-GAL4 or Gr28b.e-GAL4 (also expressing Gr28b.c; Figure 1C) caused neither attraction to nor avoidance of capsaicin.
Overlap between Gr28a and Gr66a in internal GRNs raises the question about their contribution to appetitive and/or avoidance behavior. We deemed it unlikely that these neurons were critical for appetitive behavior, since the one pair located in the DPS/VPS (expressing also Gr28b.c) is necessary for capsaicin avoidance (see above). Indeed, when VR1 was suppressed in all Gr66a/Gr28b.c neurons by means of the GAL4 suppressor GAL80, these larvae, expressing VR1 in Gr28aonly GRNs remained strongly attracted to capsaicin (Figure 2B last panel; for effective suppression, see Figure 2- figure supplement 1). Taken together, this analysis indicates that Gr28a-GAL4 is expressed in appetitive inducing neurons, while Gr28b.c-GAL4 neurons mediate avoidance behavior.
The capsaicin experiments above suggest that the two larval Gr28b.c-GAL4 GRNs mediate negative valence. Previous reports have shown that larvae avoid many bitter compounds (Apostolopoulou et al., 2016b, 2015; Choi et al., 2020, 2016; van Giesen et al., 2016), and thus, we expected that eliminating activity of Gr28b.c-GAL4 GRNs would result in loss of avoidance behavior to at least some bitter chemicals. Indeed, when Gr28b.c-GAL4 GRN activity was blocked using the inward rectifying potassium channel Kir2.1 (Baines et al., 2001; Paradis et al., 2001), larvae no longer avoided the four tested bitter compounds denatonium, quinine, lobeline and caffeine (Figure 3). A compound specific avoidance phenotype to lobeline and caffeine was observed when the Gr28b.e-GAL4 GRNs in the TOG was inactivated. These data, together with the capsaicin experiments, suggest that two pairs of GRNs, one in the TOG and one in the DPS/VPS are necessary and sufficient for avoidance of these four bitter tasting chemicals.
Gr28b.c and Gr28b.a are subunits of a taste receptor complex for denatonium
We next examined whether any of the Gr28b proteins is part of a taste receptor complex detecting any of these bitter chemicals (Figure 4). Surprisingly, only avoidance of denatonium was affected in larvae lacking the Gr28 gene cluster (ΔGr28; Figure 1B), while avoidance to quinine was somewhat reduced, albeit not significantly, and avoidance to lobeline or caffeine remained robust. In fact, avoidance to caffeine increased modestly, but significantly (Figure 4A). We then examined whether any of the Gr28 proteins was sufficient to restore denatonium avoidance by expressing individual Gr28 genes under the control of the Gr28b.c-GAL4 driver. Indeed, either Gr28b.a or Gr28b.c expression led to a full recovery of denatonium avoidance, while expression of any other Gr28b gene, or Gr28a, failed to do so (Figure 4B). This observation suggests that despite the high level of similarity between these receptors, recognition of denatonium is dependent on specific structural features present in Gr28b.a and Gr28b.c, but not in any of the other Gr28 proteins.
Since Gr66a-LexA is co-expressed in all Gr28b-GAL4 expressing GRNs, we wondered whether Gr66a is a component of the denatonium receptor. Previous work had established that Gr66a is required for caffeine avoidance, in both larvae and adult flies (Apostolopoulou et al., 2016b; Lee et al., 2009; Moon et al., 2006), which we confirmed (Figure 4-figure supplement 1). Surprisingly, avoidance of denatonium and quinine was not diminished, but increased significantly (Figure 4-figure supplement 1). Given the multimeric nature of bitter taste receptors (Sung et al., 2017), one possibility is that the absence of a Gr subunit not required for the detection of denatonium (Gr66a) could favor formation of multimeric complexes containing Gr subunits that recognize this compound (such as Gr28b.a and/or Gr28b.c).
Finally, we investigated neuronal responses in larvae expressing the Ca2+ indicator GCaMP6m in Gr28b.c-GAL4 GRNs (Figure 5). We developed a whole animal imaging preparation, whereby larvae were placed in an “imaging chamber” to minimize head movements (Figure 5A), and visualized neural activity of the Gr28b.c-GAL4 GRN in the TOG in real time (Chen et al., 2013) upon exposure to the four bitter compounds, as well as sucrose, ribose and fructose (Figure 5B to 5D). All bitter compounds elicited rapid Ca2+ increases in Gr28b.c-GAL4 GRNs, while none of the sugars did (Figure 5C and 5D). When neural activity was recorded in Gr28b.c-GAL4 GRNs of ΔGr28 homozygous mutant larvae (Figure 5E), Ca2+ responses to denatonium and quinine were severely reduced, while responses to both caffeine and lobeline were not affected. Re-expression of either Gr28b.a or Gr28b.c, but not Gr28b.b, Gr28b.d, Gr28b.e or Gr28a rescued Ca2+ response to denatonium, but not to quinine (Figure 5F and 5G). Together, these experiments identified Gr28b.c and Gr28b.a as redundant subunits of a denatonium receptor complex, a complex that does not require Gr66a or any of the other Gr28b subunits.
The Gr28 receptors comprise one of the few Gr subfamilies conserved across diverse insect species (Agnihotri et al., 2016; Engsontia and Satasook, 2021; Yu et al., 2023). Yet, they were the least characterized when compared to other conserved subfamilies, such as the sugar receptors (Gr5a, Gr61a and Gr64a-f), the carbon dioxide receptors Gr21a and Gr63a or the bitter taste receptors. The only ligands associated with the Gr28 proteins were ribonucleosides and RNA, which are appetitive nutrients essential for larvae and detected by Gr28a neurons (Mishra et al., 2018). Indeed, RNA has been found to be an appetitive taste ligand across many dipteran insects, including mosquitoes, and we showed that Gr28 homologs of both A. aegypti and A. gambiae can rescue the preference for RNA and ribose when expressed in Gr28a neurons of ΔGr28 mutant larvae (Fujii et al., 2023).
Previous studies in adult Drosophila have shown that members of conserved Gr protein families such as the carbon dioxide receptors (Gr21a and Gr63a) (Jones et al., 2007; Kwon et al., 2007) or the receptors for sweet taste encoded by the eight sugar Gr genes (Fujii et al., 2015) are largely co-expressed in one type of neuron in the fly’s taste organs. For example, with the exception of Gr5a (see below), sugar Gr genes are only expressed in a single GRN (the “sweet’ neuron) of each taste sensilla, and activation of these “sweet” neurons by sugars requires the function of at least two of the 8 sugar Gr genes (Dahanukar et al., 2007; Yavuz et al., 2014)(Fujii et al., unpublished). Similarly, the approximately 33 putative bitter taste receptors, which comprise several small conserved subfamilies (Robertson et al., 2003), as well as individual Gr genes with little overall similarity to one another, are partially co-expressed in a the bitter GRN of each taste sensilla (Weiss et al., 2011). Molecular genetic studies combined with electrophysiological recordings have shown that at least three different Gr subunits are required to constitute functional receptor complexes that can sense a bitter compound (Shim et al., 2015). We note that two rare exceptions to the heteromeric nature of taste receptor complexes exist, namely the RNA receptor Gr28a and the fructose receptor Gr43, which have been proposed to function as homomultimeric complexes (Mishra et al., 2018, 2013). Cryo-EM structural analysis of the conserved insect Olfactory receptor co-receptor (ORCO) suggests that insect odorant receptors form tetramers (Butterwick et al., 2018), and biochemical characterization and comparative modeling of BmGr9, the Bombyx mori homolog of the Drosophila Gr43a fructose receptor, supports such structures for Gr proteins as well (Morinaga et al., 2022).
Distinct functions are mediated by small set of GRNs expressing specific Gr28 subunits
Our expression analysis of the bitter taste receptor gene Gr66a and the Gr28 genes in larvae is consistent with earlier studies, despite some small variation in neuron number (Choi et al., 2016; Kwon et al., 2011), which is likely due to the use of different GAL4 driver lines and/or variability in expression levels. Importantly, all Gr28b genes are co-expressed with the bitter taste receptor Gr66a (Figure1) and probably several other putative bitter Gr genes (Kwon et al., 2011; Rist and Thum, 2017), while Gr28a is found in a largely, but not entirely, distinct set of GRNs. Whether and what kind of Gr genes might be co-expressed with Gr28 in Gr28only GRNs will require more in-depth expression studies and might shed light on other receptors involved in appetitive behaviors of larvae.
A key finding of the work presented here is the observation that Gr28aonlyand Gr28b.c neurons dictate distinct behavioral programs, the former representing an ensemble of neurons that instruct larvae to “go towards” a chemical source and consume it, while the latter do the opposite (Figure 2). This observation is reminiscent of a seminal study by Troemel and colleagues in the C. elegans chemosensory system, who reported that the valence of a chemical compound is dependent on the identity of a neuron, and not the identity of the molecular receptor the neuron expresses (Troemel et al., 1997). The number of “go-away” GRNs in Drosophila larvae co-expressing Gr28b.c and Gr66a is remarkably small, consisting of only two pairs, one in the TO and the other in the DPS/VPS. It seems likely that this is the smallest, minimal subset of neurons sufficient to induce avoidance behavior, as expression of V1R in only the TO pair (under the control of either Gr28b.a-GAL4 or Gr28b.e-GAL4) has no behavioral effect when challenged with capsaicin. The “go-to” neurons are characterized by expression of Gr28a and represent a slightly larger set of four GRN pairs (Gr28aonly GRNs). Thus, the minimal requirement to induce “go-to” and “go-away” behavior is defined by distinct sets of GRNs, and each appears to be composed of neurons located in both external as well as the internal taste organs (Figure 7). Co-expression of the RNA receptor Gr28a and in the DPS/VPS GRN essential for bitter taste (Figures 2B and 3) raises interesting questions about additional functions for Gr28a in bitter taste. We note that the sweet taste receptor Gr5a, a subunit of a multimeric trehalose receptor, is also expressed in non-sweet neurons of unknown function (Fujii et al., 2015).
Functional redundancy of taste receptors
Both behavioral analyses as well as Ca2+ imaging experiments implicate at least one Gr28b protein as an essential component of a denatonium receptor complex, as ΔGr28 larvae exhibit total loss of avoidance (Figure 4), and respective GRNs fail to elicit a response upon exposure to this chemical (Figure 3). What the precise composition of that complex is remains to be determined, but recovery of denatonium responses by expressing either Gr28b.c and/or Gr28b.a indicates that either one of these (or possibly both) is an essential subunit, in addition to other Grs expressed in this GRNs, such as Gr22a and Gr59c (Choi et al., 2020; Rist and Thum, 2017), while Gr66a is unlikely to be part of such a complex based on our behavioral analysis (Figure 4-figure supplement 1).
Since only Gr28b.a and Gr28b.c can rescue denatonium responses in Gr28b.c GRNs of ΔGr28 mutant larvae, sequence comparison between the unique N-terminal halves of the Gr28 proteins comprising the first four transmembrane domains and the extracellular loops 1 and 2 might provide insights as to possible residues important for ligand recognition. When interrogating these regions, only seven residues are identical between Gr28b.a and Gr28b.c (Table 1, Figure 6). Reducing the stringency requirement by allowing one of the remaining receptors to share the same residue, nine additional sites are identified. One experimental avenue to validate these residues as important sites contributing to denatonium binding might involve introduction of point mutations that converts respective amino acids of other Gr28b proteins into those found in Gr28b.a/Gr28b.c.
The role of Gr28b proteins in quinine detection is less clear, and the different phenotypes observed in behavioral experiments and Ca2+ imaging suggest that at least two molecular types of quinine receptors exist in larvae. Ca2+ imaging experiments implicate a role for multiple Gr28b subunits in a quinine receptor complex in the TOG neuron, since single Gr28b genes cannot restore the loss of quinine response in ΔGr28 larvae (Figure 5E and 5G). However, because ΔGr28 mutant larvae still avoid quinine (Figure 4), at least one Gr28b independent receptor must be exist in one or several other GRNs, one of which is likely the DPS/VPS, since expression of Kir2.1 in that neuron, along with the TOG neuron, totally abolishes quinine avoidance (Figure 3). We note that functional redundancy is also observed in sweet taste receptors, where we found that different combinations of sugar Gr genes can restore responses to the same sugar when expressed in tarsal sweet GRNs of sugar blind flies (Fujii et al. unpublished).
Materials and methods
Key resources table
Flies were maintained on standard corn meal food in plastic vials under a 12 h light/dark cycle at 25°C. The w1118 strain (Bloomington Drosophila Stock Center, number 3605) was used as a wild-type control. Fly strains used: Gr28a-GAL4(SF36S) for Figure 1 and Figure 2B, and SF36E1 for Figure 2B and Figure 2-figure supplement 1, Gr28b.a-GAL4(NT42aC51a), Gr28b.c-GAL4(NT21B1) (Thorne and Amrein, 2008); Gr28b.e-GAL4(Gr28a3AII) and Gr66a-GAL4 (Scott et al., 2001); ΔGr28(54B3) (Mishra et al., 2018); UAS-Gr28a, UAS-Gr28b.a, UAS-Gr28b.b, UAS-Gr28b.c, UAS-Gr28b.d and UAS-Gr28b.e (Ni et al., 2013); Gr43aGAL4 (Miyamoto et al., 2012); Gr66a-LexA and lexAop-GAL80 (Thistle et al., 2012); UAS-VR1E600K (Marella et al., 2006); UAS-Kir2.1-GFP (Baines et al., 2001; Paradis et al., 2001); lexAop-rCD2:GFP (Lai and Lee, 2006), UAS-GCaMP6m, UAS-mCD8:RFP and Gr66aex83 (Bloomington Drosophila Stock Center, numbers 42748, 32220 and 35528); Gr28b.c-LexA(#8)
Caffeine (Cat No. C0750), capsaicin (Cat No. M2028), denatonium benzoate (Cat No. D5765) and lobeline hydrochloride (Cat No. 141879), D-(-)-ribose (Cat No. R7500) and quinine hydrochloride dihydrate (Cat No. Q1125) were purchased from Millipore-Sigma, with a purity of > 95%. Fructose (Cat No. F1092) and agarose (Cat. No. 20-102) were purchased from Spectrum chemical and Apexbio, respectively. Sucrose (Mfr. No. 8360-06) and charcoal (Cat No. 1560-01) were purchased from Macron fine chemicals and J.T. Baker, respectively. A stock solution for capsaicin (20 mM) was prepared in 70% ethanol and stored at 4° C protected from light for up to one year. Stock solutions for bitter chemicals were prepared in Millipore Q water and stored at -20° C. Stock solutions for sugars were prepared in Millipore Q water and stored at 4° C for up to one month. A stock solution for ribose was treated with charcoal (10% of the weight of ribose used for stock solution) overnight at 4° C and sterile-filtrated (0.45 μm) to remove unrelated odor. Stock solutions were diluted to the final concentration using Millipore Q water prior to each experiment.
Immunofluorescence of larval heads was performed based on the protocol described in Croset and colleagues (Croset et al., 2016) with minor modification. Heads of third instar larvae were dissected using microscissors in phosphate buffered saline (PBS) and immediately fixed in PBS with 4% paraformaldehyde for 1 hour at 4 °C. They were washed six times in washing buffer (PBS with 0.1% Triton X-100) for 20 min and blocked for 1 h in washing buffer containing 5% heat-inactivated goat serum (SouthernBiotech, Cat. No. 0060-01), followed by incubation with the primary antibodies (rabbit anti–GFP, 1:1,000 dilution; rat anti–mCD8, 1:200 dilution, ThermoFisher Scientific) at 4°C overnight. The next day, heads were washed six times for 20 min in washing buffer and blocked in washing buffer containing 5% heat-inactivated goat serum for 1 hour, followed by incubation with the secondary antibodies (goat anti rabbit ALEXA 488, 1:500 dilution, ThermoFisher Scientific; goat anti–rat Cy3, 1:300 dilution, Jackson Immunoresearch Laboratories Inc.) at 4°C overnight. Finally, heads were washed six times in washing buffer for 20 min each at room temperature under gentle agitation. Heads were then mounted with VectaShield (Vector Lab, Cat No. H-1200) on a microscope slide and images were obtained using a Nikon A1R confocal microscope system. Adobe photoshop 2022 was used further to process images.
Larval two-choice preference assay
Two-choice preference assay of larvae was conducted as described in Mishra et al. (Mishra et al., 2013) with minor modifications. Flies were placed on standard corn meal food in plastic vials and allowed to lay eggs for 24 h under a 12 h light/dark cycle at 25°C. Flies were removed from food vials and feeding-stage third-instar larvae were collected. Agarose food dishes for two-choice preference assay were prepared just prior each experiment as follows: Petri dishes (60 x 15 mm, Falcon, Cat. No. REF353004) with two halves marked on the bottom were filled with melted plain 1% agarose or 1% agarose containing 1.75% ethanol (for capsaicin preference). After the agarose solidified, one half was removed and replaced with 1% agarose solution containing taste ligands (capsaicin or bitter compound). For each experiment, 15 larvae from food vials were briefly rinsed twice with Millipore Q water and placed along the middle separating pure and ligand containing agarose. After 16 min, images were taken for record keeping and used to calculate larval preference indices. Larvae that crawled onto the wall of a dish or dug in the agarose were excluded. The preference index (P.I.) was calculated as follow: PI = (Ntastant - Nplain) / NTotal, whereby N is the number of larvae in the tastant sector, the plain agarose sector and the total number, respectively. Positive values indicate a preference for capsaicin or bitter compound while negative values indicate repulsion (avoidance).
Calcium imaging was performed in Gr28b.c GRNs expressed in the terminal organ of feeding-stage, third instar larvae, reared as described for the larval two-choice preference assay. For each experiment, larvae from food vials were briefly rinsed twice with Millipore Q water and were mounted dorsally on a large microscope cover glass (24 x 50 mm, VWR, Cat. No. 16004-098) using double-sided scotch tape and covered with a small microscope cover glass (12CIR-1, Thermo Fisher Scientific, Cat. No. 1254580). Millipore Q water (40 µl) was applied to the tip of the larval head, and the preparation was placed on the stage of a Nikon eclipse Ti inverted microscope. Images were obtained every 500 ms, starting 15 s before application and ending 105 s after ligand application. Each recording was initiated by applying water (40μl) to set a baseline. The first ligand solution (40 μl of bitter chemical or sugar) was applied thereafter, followed by 5 washes with carrier (100 μl of water). After a 3-minute pause to allow the preparation to recalibrate, a second ligand solution (40 μl bitter chemical or sugar) was applied. To assure validity in experiments with Gr28 mutants and rescues, each recording was concluded with application of caffeine, and recordings were included only if caffeine generated a positive response. Baseline fluorescence, which was determined from the average of five frame measurements from a region next to the cell immediately before ligand application, was subtracted from the actual measurements. ΔF/F (%) = (fluorescence light intensity of the cell body – baseline/baseline) x 100. ΔF/F (max %) is the maximum value within 40 seconds after ligand application.
Generation of transgenic Gr28b.c-LexA flies
To generate the Gr28b.c-LexA driver, a 1.3 kb DNA fragment immediately upstream of the Gr28b.c start codon was amplified from w1118 flies using a forward (5′-AATCTAGGTACCCCGGCTGCTCGTCTCCCTGGATGT-3′) and a reverse (5′- CGTCAAACTAGTGACCGCTTCGTTTGAGCTTCAACC-3′) primer. Acc65I and SpeI sites included in the primer sequence (underlined) were incorporated such that the amplified fragment was amenable to directional cloning into the LexA vector CMC105 (Larsson et al., 2004). The clone chosen was confirmed by DNA sequence analysis. Transgenic flies were generated by standard P-element transformation of w1118 embryos (Rainbow Transgenic Flies Inc., Camarillo, CA).
Statistical analyses were conducted using Prism software 9.5.1 (GraphPad Software). Larval two-choice preference assay and Ca2+ imaging data were analyzed for normal distribution using D’Agostino-Pearson omnibus and Shapiro-Wilk normality tests. When groups did not meet the assumption for normal distribution, nonparametric statistics was used. For comparison between multiple groups, one-way ANOVA or Kruskal-Wallis test by ranks (nonparametric one-way ANOVA) was performed to test for difference of mean or rank distribution. As a post hoc test, Bonferroni’s or Dunn’s (nonparametric) multiple comparison tests were employed to compare two specific groups. One-way ANOVA with Bonferroni’s multiple comparison tests were used in Figure 2. Kruskal-Wallis test by ranks with Dunn’s multiple comparison tests were used in Figures 3, 4B and 5F and 5G). For comparison between two groups, Mann-Whitney U test (nonparametric t test, Figures 4A, 5D, 5E, Figure 2-figure supplement 1B and Figure 4-figure supplement 1) with two-tailed P value were used to compare means of two groups. Sample size for larval two-choice preference assays and Ca2+ imaging experiments were based on Mishra et al (Mishra et al., 2018).
We thank Tetsuya Miyamoto, Shinsuke Fujii and Sheida Hedjazi for valuable suggestions throughout the duration of this project and Raquel Sitcheran for comments on the manuscript. We are grateful to Paul Garrity for the UAS-Gr28 reporter strains and the Bloomington stock center for numerous Drosophila strains. This work was supported by NIH grants1 R01 DC018403, R21 DC015327 and R01GMDC05606 to H. Amrein. Drs. Ahn and Amrein conceived the experiments, Dr. Ahn conducted all experiments, and Dr. Amrein wrote the paper.
Source data files
Figure 2-Source Data 1. Taste preference assay of larvae expressing VR1 in different Gr GRNs for 0.1 mM capsaicin. Taste preference assay of larvae with expression of VR1 in Gr28aonly GRNs using lexAop-GAL80 under control of Gr66a-LexA for 0.1 mM capsaicin.
Figure 3-Source Data 1. Taste preference assay for bitter compounds of larvae with inactivated Gr28b.c or Gr28b.e GRNs using expression of UAS-Kir2.1.
Figure 4-Source Data 1. (A) Taste preference assay of wild type and Gr28 mutant larvae for bitter compounds. (B) Taste preference assay of Gr28 mutant larvae for denatonium expressing single Gr28 genes in Gr28b.c GRNs.
Figure 5-Source Data 1. (D) Ca2+ responses of Gr28b.c GRNs in the TOG to bitter compounds and sugars. (E) Ca2+ responses of Gr28 mutant larvae to bitter compounds. (F&G) Ca2+ responses of Gr28 mutant larvae to denatonium (F) or quinine (G) expressing single Gr28 genes in Gr28b.c GRNs.
Figure 2-figure supplement 1-Source Data 1A & 1B. (B) Quantification of GFP positive GRNs recorded from images of Gr28a GRNs expressing UAS-GCaMP6m (A).
Figure 4-figure supplement 1-Source Data 1. Taste preference assay of Gr66a mutant larvae for bitter compounds.
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