Introduction

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 gustatory system in Drosophila is characterized by several insect specific features with regard to anatomy and the molecular nature of taste receptors. For example, taste cells are primary sensory neurons, with dendritic projections that express taste receptors, while long axons project that 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 (Joseph and Carlson, 2015)(Amrein, 2016)(Scott, 2018). Similarly, Drosophila larvae have numerous structures located both on the head surface as well as internally along the larval pharynx, that assess chemical compounds prior and during ingestion, respectively (Kwon et al., 2011)(Apostolopoulou et al., 2015)(Rist and Thum, 2017). Like most insects, Drosophila employs taste receptors encoded by two major gene families, the Gustatory receptor (Gr) and the Ionotropic Receptor (IR) genes, to sense all chemicals, such as appetitive food compounds, noxious and toxic chemicals as well as pheromones. Both Gr and IR based receptors are thought of form complexes composed of several different subunits, partial composition of which is known for only a few Gr-based taste receptors for sugars and bitt er compounds (Kwon et al., 2007)(Jiao et al., 2008)(Fujii et al., 2015)(Shim et al., 2015)(Gonzales et al., unpublished), as well as gaseous carbon dioxide (Jones et al., 2007) and even fewer IR-based fatty acid and carboxylic acid receptors (Ahn et al., 2017)(Tauber et al., 2017)(Chen and Amrein, 2017). Interestingly, at least some GRNs express members of both families, although no evidence for receptor complexes consisting of both IR and Gr subunits has been reported to date.

The Gr genes represent the largest taste receptor gene family in insects. In D. melanogaster, it comprises 60 genes predicted to encode 68 proteins, expression of which has been characterized (Kwon et al., 2011)(Weiss et al., 2011)(Ling et al., 2014)(Choi et al., 2016)(Rist and Thum, 2017). Several Gr genes have been functionally characterized in adult flies using genetic mutations, combined with either electrophysiological recordings, Ca²⁺ imaging studies and behavioral analyses (reviewed by Montell, 2021) but only a few have been studied in larvae (Mishra et al., 2013)(Apostolopoulou et al., 2016)(Choi et al., 2016)(Mishra et al., 2018)(Choi et al., 2020). The Gr28 gene subfamily is of particular interest for a number of reasons: First, it is one of the most conserved Gr subfamily, homologs of which can be found across all insect families and even more distant arthropods (Eyun et al., 2017)(Suzuki et al., 2018)(Fujii et al., 2023). Second, the Gr28 genes are expressed not only in the gustatory system, but 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 (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 Gr28bd 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 gustatory function for any Gr28 gene in larvae thus far is sensing of ribonucleosides and RNA in larvae (Mishra et al., 2018), which was recently established as a novel appetitive taste quality common to a broad range of dipteran insects (Fujii et al., 2023).

Here, we present a functional characterization of the Gr28 genes and the respective GRNs in Drosophila larvae. Four of the six Gr28 genes (Gr28a, Gr28ba, Gr28bc and Gr28be) are expressed in the larval taste system, subdivided into two, functionally distinct neuronal ensembles. The first such ensemble is defined by expression of Gr28bc and comprises four neurons, two of which also express Gr28ba, Gr28bc, Gr28be and the bitter core receptor gene Gr66a, while the other two neurons express only Gr28bc, Gr28a and Gr66a. The second ensemble is characterized by 12 GRNs that express Gr28a, including the pair of GRNs expressing the Gr28b genes. When the mammalian Vanilloid Receptor 1 (VR1) was expressed under the control of specific GAL4 drivers, we found that Gr28a and Gr28bc neurons mediate opposing taste behavior in the presence of capsaicin, the ligand for VR1. Specifically, Gr28a:VR1 larvae show strong attraction for capsaicin, while Gr28bc:VR1 larvae show strong avoidance of capsaicin. Furthermore, inactivation experiments reveal that the Gr28bc GRNs are necessary to sense bitter compounds, specifically denatonium, quinine, lobeline, and caffeine. However, Gr28 mutant larvae fail to avoid only denatonium, but none of the other bitter compounds, a phenotype rescued by expression of Gr28bc or Gr28ba. Thus, to our knowledge, the related Gr28 genes represent the first subfamily where members mediate functionally opposing taste behaviors.

Results

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 organized as paired ganglia, the Dorsal, Terminal and Ventral Organ Ganglia (DOGs, TOGs and VOGs) that house the GRN cell bodies, each neuron projecting a dendrite to the sensory organ structures (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 taste neurons. For clarity, numbers below refer to neurons on one side of the bilaterally symmetrical structures of all organs, thus the total neuron number in a larva is double that number. The presumptive GRNs located in the DOG fall into two distinct groups, based on their dendritic projections: 11 presumptive GRNs, four of which were shown to express Gr genes (Apostolopoulou et al., 2015), extend dendritic projections to the base of the DO, while 7 neurons project dendrites to the TO (the dorsolateral group, Figure 1A)(Kwon et al., 2011). The TOG contains 30 neurons, all sending dendrites to 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 pass 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 (Stewart et al., 2015)(Rist and Thum, 2017)(Sánchez-Alcañiz et al., 2018).

Figure 1.

Our initial expression analysis revealed that four of the six Gr28 genes (Gr28a, Gr28ba, Gr28bc and Gr28be) are 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). To further delineate the putative chemosensory roles of these genes, we performed a more detailed co-expression analysis between Gr28 genes and with respect to the bitter receptor gene Gr66a, using the bimodal expression systems GAL4/UAS to label Gr28 expressing neurons and LexA/lexAop to mark Gr66a neurons (Figure 1B and 1C; note that the neuron numbers below refer to the number present in one of each paired ganglion). In the TOG, we found expression of all four Gr28 genes, along with that Gr66a, which was expressed in 3 or 4 neurons (one neuron exhibits often stronger expression of Gr66a than the two or three remaining neurons). This number slightly smaller than the 6 neurons reported using a distinct Gr66a-GAL4 driver (Kwon et al., 2011). Gr28ba, Gr28bc and Gr28be are co-expressed in a single neuron with Gr66a (Figure 1B). In contrast, Gr28a is expressed in a distinct TOG neuron than Gr66a (Figure 1B), and by extension, in a different neuron than the co-expressed Gr28b genes, which we independently confirmed using a Gr28bc-QF driver (Figure 1-figure supplement 1). In the DOG, we find only a single Gr28a neuron, while none of the Gr28b genes, or Gr66a, is expressed there (Figure 1B). Expression in the internal sensory organs was similarly complex. Gr66a was found in one neuron in the DPS/VPS, co-expressed with Gr28bc as well as Gr28a (Figure 1C). None of the Gr28b genes is expressed in the PPS, but two neurons express Gr28a, one of which also expresses Gr66a (Figure 1C). In summary, the internal sensory organs can be subdivided in three distinct groups: Gr66a/Gr28b/Gr28a positive neurons, Gr66a/Gr28a positive neurons and Gr28a^only neurons, which contrasts expression in the external sensory organs where Gr28a and the Gr28b genes are mutually exclusive.

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). Specifically, larvae homozygous for a Gr28 null allele, a deletion of the entire Gr28a gene and most of the coding region of all Gr28b genes (ΔGr28), lose their ability to sense these compounds, a phenotype that is restored when single UAS-Gr28 genes are expressed in Gr28a neurons using Gr28a-GAL4. This observation and 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, Gr28ba-GAL4, Gr28bc-GAL4 and Gr28be-GAL4) in larvae and tested their response to capsaicin using the two-choice preference assay. Additionally, we expressed UAS-VR1 in bitter neurons as well as sweet neurons using respective GAL4 drivers (Gr66a-GAL4 and Gr43a^GAL4) (Scott et al., 2001) (Miyamoto et al., 2012). Consistent with the observation in adults (Marella et al., 2006), w¹¹¹⁸ control larvae and larvae carrying UAS-VR1 or Gr-GAL4 genes alone were unresponsive to 0.1 mM or 0.5 mM capsaicin (Figure 2A). However, larvae expressing UAS-VR1 under the control of Gr28a-GAL4 or Gr43a-GAL4 showed strong appetitive responses to both capsaicin concentrations (Figure 2A). In contrast, larvae expressing UAS-VR1 under the control of Gr28bc-GAL4 or Gr66a-GAL4 showed robust avoidance behavior to both concentrations of capsaicin. Lastly, expression of UAS-VR1 with the Gr28ba-GAL4 or Gr28be-GAL4 driver, which is co-expressed in a single TO neuron with Gr28bc (Figure 1B), did cause neither attraction to nor avoidance of capsaicin.

Figure 2.

Expression difference between Gr28bc and Gr28ba/be is a single neuron in the DPS/VPS (Figure 1C), a neuron required to mediate avoidance of capsaicin in Gr28bc-GAL4, UAS-VR1 larvae. Even though this GRN also expresses Gr28a, it appears unlikely that it is necessary to mediate appetitive behavior as well. Indeed, we could further restrict the necessary set of appetitive neurons to four Gr28a positive neurons (Gr28a^onlyGRNs), as suppression of VR1 expression in Gr66a GRNs (one being the Gr28bc expressing GRN in the DPS/VPS) did not affect capsaicin preference (Figure 2B). We further note that activation of the two Gr28bc neurons by capsaicin drives repulsion to the same degree as when all seven Gr66a expressing neurons are activated. Taken together, this analysis indicates that Gr28a-GAL4 is expressed in appetitive inducing neurons, while Gr28bc-GAL4 neurons mediate avoidance behavior.

Wild type larvae, like adult flies, avoid bitter compounds (Apostolopoulou et al., 2015) (Apostolopoulou et al., 2016) (Choi et al., 2016) (van Giesen et al., 2016) (Choi et al., 2020). If Gr28bc neurons are indeed central to the avoidance of these chemicals in larvae, we expect that eliminating their function would result in loss of larval avoidance behavior to bitter chemicals. Thus, we inactivated neuronal subsets using the potassium inward rectifying channel Kir2.1 (Baines et al., 2001) (Paradis et al., 2001). In contrast to larvae with an intact taste neuron repertoire, larvae in which two Gr28bc neurons were inactivated no longer avoided any of these bitter compounds (Figure 3). A more subtle phenotype was observed when the single Gr28be neurons were inactivated, eliminating lobeline and caffeine avoidance, but leaving denatonium and quinine repulsion intact, a finding consistent with previous studies (Choi et al., 2020). These data suggest that two pairs of neurons, one in the TO and one in the D/VPS are necessary and sufficient for avoidance of all four bitter tasting chemicals.

Figure 3.

Gr28bc and Gr28ba are subunits of a taste receptor complex for denatonium

We examined whether any of the Gr28 proteins is part of a taste receptor complex detecting these bitter chemicals (Figure 4). Surprisingly, only avoidance of denatonium was affected in larvae without any functional Gr28 gene (ΔGr28), while responses to quinine, lobeline or caffeine was not significantly affected. Moreover, avoidance to caffeine increased significantly (Figure 4A). We next tested which Gr28 protein is necessary for denatonium avoidance and expressed individual Gr28 genes under the control of the Gr28bc-GAL4 driver. Only when either Gr28ba or Gr28bc was expressed in these neurons, denatonium avoidance was fully recovered, whereas expression of Gr28bb, Gr28bd, Gr28be or Gr28a failed to do so (Figure 4B). This observation indicates that despite the high level of similarity between these receptors, recognition of denatonium is dependent on specific Gr28b subunits, features that are present in Gr28ba and Gr28bc, but not any of the other Gr28 proteins.

Figure 4.

Previous work had established that Gr66a is required for caffeine avoidance, in both larvae and adult flies (Moon et al., 2006)(Lee et al., 2009)(Apostolopoulou et al., 2016). We confirmed this, as Gr66a mutant larvae show little if any avoidance of caffeine, while avoidance of lobeline was not affected (Figure 4-figure supplement 1). Somewhat surprisingly, avoidance of denatonium and quinine increased significantly. This is likely a result of the heteromeric composition of bitter taste receptor complexes, whereby absence of Gr66a allows Grs that tuned to denatonium (such as Gr28bc or Gr28ba) and quinine be incorporated into multimeric receptor complexes.

Finally, we investigated neuronal responses in larvae expressing the Ca²⁺ indicator GCaMP6m in Gr28bc-GAL4 GRNs (Figure 5). We developed a whole larvae imaging preparation, whereby larvae were placed in an “imaging chamber” to minimize head movements (Figure 5A), and visualized neural activity 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 Gr28bc GRNs in the TO, while none of the sugars did (Figure 5C and 5D). When neural activity was recorded in ΔGr28 homozygous mutant larvae (Figure 5E), cellular responses to denatonium and quinine were severely reduced, while responses to both caffeine and lobeline were not affected. Consistent with our behavioral analysis (Figure 4B), re-expression of either Gr28ba or Gr28bc, but not Gr28bb, Gr28bd, Gr28be or Gr28a rescued cellular response to denatonium, but not to quinine (Figure 5E). Together, these experiments identified Gr28bc and Gr28ba as redundant subunit of a denatonium receptor complex, a complex that does not require Gr66a or any of the other Gr28b subunits.

Figure 5.

Discussion

The Gr28 receptors comprise one of the few Gr subfamilies conserved across diverse insect species (Engsontia and Satasook, 2021)(Agnihotri et al., 2016)(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 for larvae and are mediated 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). Here, we established a precedent for members of the same subfamily for mediating both appetitive and repulsive behavior and being expressed in largely non-overlapping sets of GRNs. In flies, members of other conserved Gr subfamilies are either precisely (Gr21a and Gr63a)(Jones et al., 2007)(Kwon et al., 2007), or largely (sugar Gr genes) (Fujii et al., 2015) co-expressed in one type of GRNs, forming multimeric receptors that mediate a single type of behavior.

Our expression analysis in larvae is consistent with earlier reports, despite some variation in number of neurons expressing Gr66a and Gr28b (Kwon et al., 2011)(Choi et al., 2016), which is likely due to the use of different GAL4 drivers and/or variability in expression. Importantly, all Gr28b genes are co-expressed with the bitter marker Gr66a (Figure1), while Gr28a is found in a largely, but not entirely distinct set of GRNs. Based on our VR1-capsaicin experiments, the GRNs sharing expression of both Gr28a and Gr66a (and by extension some Gr28b genes) are critical for avoidance, and not required for appetitive behavior (Figure 2).

Distinct functions of small set of GRNs expressing specific Gr28 subunits

A key finding presented of this study is the observation that Gr28a^only and Gr28bc neurons dictate distinct behavioral programs, the former representing an ensemble of neurons that instruct a larvae to “go towards” chemical source and consume it, while the latter do the opposite (Figure 2). The set of “go-away” GRNs co-expressing Gr28bc 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 for avoidance, as expression of V1R in only the TO pair (under the control of either Gr28ba-GAL4 or Gr28be-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 (Gr28a^only only GRNs). Thus, we propose that the minimal requirement to induce “go-to” and “go-away” behavior is defined by distinct sets of GRNs, and that each appears to be composed of neurons located in both external as well as the internal taste organs (Figure 6).

Figure 6

Co-expression of a subunit for the bitter taste receptors Gr28bc/Gr28ba and Gr28a, a receptor for RNA, a nutrient essential for larval growth (Mishra et al., 2018) in bitter taste neurons raises interesting questions about the function of Gr28a. As these neurons are necessary for repulsion, we propose that Gr28a might serve as a subunit along with other Grs (possibly Gr28bc and/or Gr66a) in heteromeric bitter taste receptor complexes, detecting yet other bitter compounds. In “go-to”, Gr28a^only GRNs, Gr28a is likely to form a homomultimeric receptor, since its expression in fructose sensing GRNs confers RNA recognition to these neurons, both in cellular and behavioral assays (Mishra et al., 2018). We note that the sweet taste receptor Gr5a, a subunit of a multimeric receptor in sweet taste GRNs in the labial palps of flies is also expressed in non-sweet neurons of unknown function (Fujii et al., 2015). Finally, while Gr28bc was shown to be required for detecting and avoiding 5% saponin in flies (Sang et al., 2019), larvae showed no behavioral response to 0.1% of saponin (Choi et al., 2016)(Choi et al., 2020), a finding we independently confirmed using both concentration of saponin (data not shown).

Mapping ligand specificity for denatonium

Only Gr28ba and Gr28bc can rescue responses to denatonium when expressed in Gr28bc GRNs of ΔGr28 mutant larvae. Thus, sequence comparison between the unique N-terminal halves of the Gr28 proteins might provide insights as to possible residues important for denatonium recognition/binding, especially residues located in extracellular loops (EL) and transmembrane (TM) regions. When interrogating TM domains 1 to 4 and EL1 and EL2, only seven residues are identical between Gr28ba and Gr28bc (Table 1, Figure 7). Reducing the stringency requirement by allowing one of the remaining receptors to share the same residue, nine additional sites are identified. Interestingly, five of these sixteen amino acid residues map to four of the nine most probable binding pocket as predicted by Prankweb (https://prankweb.cz/), with three residues part of the pocket with the highest probability score (Table 2, Figure 8). Thus, targeted substitutions of single amino acids residing in predicted ligand binding pockets in other Gr28 proteins and testing such “chimera” provides a theoretical framework for practical experiments using either behavior analysis or Ca2+ imaging to precisely define the specific requirement for denatonium specificity. Additional complementary approaches can be taken to identify the critical residues in the predicted binding pockets of other members of this family once their ligands have been identified. Thus, the Gr28 proteins might serve as model to experimentally validate the predictions created by these powerful algorithms.

Figure 7

Figure 8.

Table 1.

Table 2.

Materials and methods

Key resources table

Drosophila Stocks

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 immunostaining and Figure 2B, and SF36B1 for all other experiments), Gr28ba-GAL4(NT42aC51a), Gr28bc-GAL4(NT21B1) (Thorne and Amrein, 2008); Gr28be-GAL4(Gr28a3AII) and Gr66a-GAL4 (Scott et al., 2001); ΔGr28(54B3) (Mishra et al., 2018); UAS-Gr28a, UAS-Gr28ba, UAS-Gr28bb, UAS-Gr28bc, UAS-Gr28bd and UAS-Gr28be (Ni et al., 2013); Gr43a^GAL4 (Miyamoto et al., 2012); Gr66a-LexA (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 Gr66a^ex83(Bloomington Drosophila Stock Center, numbers 42748, 32220 and 35528)

Chemicals

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

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. Heads 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. 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. Heads were incubated 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. Heads were washed six times in washing buffer for 20 min. Washes were carried out at room temperature under gentle agitation. Heads were 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 modification. 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. All dishes for two-choice preference assay were prepared just prior each experiment. 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, photos 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 = (N_tastant - N_plain) / N_Total, whereby N is the number of larvae in the tastant sector, the plain agarose sector and the total number on the plate. Positive values indicate a preference for capsaicin or bitter compound while negative values indicate repulsion (avoidance).

Calcium imaging

Calcium imaging was performed in Gr28bc 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 Gr28bc-LexA flies

To generate the Gr28bc-LexA driver, a 1.3 kb DNA fragment immediately upstream of the Gr28bc start codon was amplified from w¹¹¹⁸flies 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 analysis

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 and Figure 4-figure supplement 1) with two-tailed P value was 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).

Acknowledgements

We thank Tetsuya Miyamoto, Shinsuke Fujii and Sheida Hedjazi for valuable suggestions throughout the duration of this project. 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. (A) Taste preference assay of larvae expressing VR1 in different Gr28 GRNs for 0.1 mM (top) or 0.5 mM (bottom) capsaicin. (B) Taste preference assay of larvae with expression of VR1 in Gr28a^only GRNs using lexAop-GAL80 under control of Gr66a-LexA.

Figure 3-Source Data 1. Taste preference assay for bitter compounds of larvae with inactivated Gr28bc or Gr28be 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 Gr28bc GRNs.

Figure 5-Source Data 1. (D) Ca²⁺ responses of Gr28bc GRNs in the TO 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 Gr28bc GRNs.

Figure 4-figure supplement 1-Source Data 1. Taste preference assay of Gr66a mutant larvae for bitter compounds.