The Drosophila IR20a Integrates L-arginine and Salt Signals via Distinct Subunit Assemblies

Reviewer #1 (Public review):

Summary:

The authors investigated the function of a Drosophila chemosensory receptor, IR20a, using genetics, neuronal histology, calcium imaging (in vivo and in cultured cells), and behavioral approaches. They provide evidence that this receptor functions in the detection of the amino acid arginine and of low salt (NaCl) concentrations, functioning in different combinations with "co-receptor" IRs, IR25a and IR76b.

Strengths:

The experiments are generally very well-performed and clearly presented, using established methodology. While, unsurprisingly, some puzzles remain (mentioned below), the work provides one of the clearest lines of evidence for the combinatorial coding of sensory information at the periphery through the combined action of distinct sets of chemosensory IRs.

As taste neurons have long been recognized to express many different combinations of IRs and Gustatory Receptors (GRs), this study will be of interest to chemosensory biologists in general, particularly those studying invertebrate model systems (though co-expression of different families of taste receptors is a feature of mammalian taste cells).

The precise molecular mechanisms remain unclear: there is no direct evidence here for protein complex formation (though this is likely), the stoichiometry of such complexes, or how subunits interact to confer or suppress sensory sensitivity. Nevertheless, these receptors, and the authors' success in reconstituting functionality in cultured cells, might make these a powerful model to explore such questions in the future.

Weaknesses:

Given the particular interest of the data from the heterologous reconstitution in cultured cells, the authors should be quite explicit about the nature of the quantification of the S2 cell responses. It is unclear whether the cited "n" refers to numbers of cells or something else, and whether all or only a fraction of (transfected) cells gave responses.

There has been some prior work on the context-specific role of IR76b in amino acid-sensing and salt sensing by Ganguly and colleagues (Cell Reports 2017), who also implicated (weakly) a contribution of IR20a in contributing to the amino acid-sensing role. In that work, the authors focussed principally on the labellum and used electrophysiology rather than calcium imaging. The present manuscript appears rather dismissive of the earlier results (only mentioning them in the Discussion), and the authors could be a bit more generous about what was previously determined, where they have confirmed previous findings, where their results diverge, and why this might be. Similarly, the original functional analysis of IR76b (Zhang Science 2013) argued this was a low-salt sensor by itself, which is at least partially corroborated here; it remains unclear how this role relates to the low-salt detecting function of a potential complex of IR20a/IR25a/IR76b. It would be useful to have a summary model of the possible variety of complexes of IRs in different types of sensory neurons, as supported by the results in this and previous studies.

The discord between the lack of requirement for IR20a for physiological responses to arginine in tarsi versus the necessity for behavioral responses is puzzling (though might reflect a labellar role for IR20a). There appears to be a trend of a decrease in calcium signal in tarsi to 100 mM arginine, which is the highest concentration tested (Figure 2A, C). Would a statistically significant decrease be observed with lower arginine concentrations? (A more substantial experiment would be to perform calcium imaging in the labellar IR20a neurons, or their axonal projections in the SEZ; this is not necessary, but the authors should at least acknowledge that their imaging of tarsal responses, while convenient, only examines a tiny fraction of the entire IR20a neuron population.

The authors argue for synergistic responses to arginine and NaCl mediated by IR20a/IR25a. It's not clear to me to what extent there is synergism. In Figure 5A, 10 mM arginine or 10 mM NaCl individually lead to c.30-40% PER, and then when both are presented together in the "Mix" (presumably both compounds at 10 mM?), PER rises to c.60%. Is this really synergism, or rather simple additivity of behavioral responses to two attractive compounds? The authors could discuss this more thoroughly. Similarly, in Figure 5G the authors show that 50 mM arginine does not evoke a significant response in S2 cells expressing IR25a/IR20a, but in Figure 4 it would seem likely that a 50 mM dose would produce a significant response (the response to 25 mM arginine in Figure 4F is already elevated above the control, albeit not statistically significant). Is this just a batch effect of the experiments performed at different times (so they are not directly comparable)?

The legend title to Figure 6 implies cooperation between tonic and state-modulated pathways, but I don't see specific evidence for "cooperation". Rather, as in the results text, they seem to work in parallel, so this analysis seems slightly peripheral to the main focus of the manuscript. It's ultimately unclear how the IR20a/IR76b/IR25a low salt sensor and the sensor containing IR56b functionally interact at the behavioral level. Here, a graphical summary, as mentioned above, of the different salt sensing neurons, the receptors they use, and the behaviors they control could be useful to establish the current knowledge and highlight open questions for the future.

Reviewer #2 (Public review):

Summary:

This study identifies IR20a-expressing gustatory neurons in Drosophila as a multimodal sensory population integrating amino acid (arginine) and low-salt signals through combinatorial IR20a/IR25a/IR76b receptor assemblies. The proposed model of peripheral-level signal integration and synergistic enhancement of feeding preference is potentially significant, as it expands current understanding of gustatory coding beyond single-modality labeled lines.

Strengths:

Overall, the findings are conceptually interesting and suggest a novel framework for multimodal taste integration, but some mechanistic interpretations remain incompletely supported by direct evidence.

Weaknesses:

(1) Although the authors demonstrate co-expression of IR20a, IR25a, and IR76b in the same GRN population, this evidence is insufficient to support the proposed model of distinct receptors coexisting within individual neurons. Additional molecular or structural data would be required to distinguish whether these subunits assemble into complexes.

(2) Given that IR76b has already been established as a sodium/salt sensing channel, the novelty of this study relies on the proposed role of IR20a in conferring multimodal integration and synergy. However, it remains unclear whether this represents a fundamentally new sensory mechanism or a re-interpretation of known IR76b-dependent salt responses in a different neuronal context.

(3) Line 127:
-The statement that there is no overlap between IR20a-GAL4 and GR64f-LexA or GR66a-LexA is not sufficiently supported by the presented imaging data. In particular, the resolution and clarity of the confocal images in Figure 1 appear suboptimal, making it difficult to confidently assess co-localization. The authors are encouraged to provide higher-resolution images or additional quantitative co-localization analysis to substantiate this conclusion.
-In addition, the images shown in Figure 1 F1-F2 suggest possible partial overlap between IR20a and GR66a signals, which appears inconsistent with the authors' statement of no co-expression. This discrepancy should be clarified.

(4) Lines 138-141:
There appears to be a discrepancy between imaging and behavioral data: IR20a is reported as dispensable for arginine-evoked neural responses, yet IR20a mutants show significantly reduced attraction to arginine in behavioral assays. The authors should clarify how behavioral deficits arise in the absence of detectable changes in calcium imaging,

(5) The manuscript proposes that IR20a functions in combination with IR25a to mediate multimodal detection of arginine and low NaCl. However, the specific role of IR25a in this context remains unclear.

(6) The authors report that co-expression of IR20a and IR25a confers synergistic responses to combined arginine and NaCl stimulation, whereas the inclusion of IR76b abolishes this response (Figures 5E-K). This is an intriguing and potentially important finding; however, the mechanistic basis for this suppression is not clearly explained.

(7) The authors propose that IR56b mediates state-dependent modulation of low-salt preference. However, the current data do not clearly distinguish whether IR56b acts as a real nutrient state sensor or just functions as a downstream modulatory component within a broader feeding circuit. Additional evidence linking IR56b activity changes to upstream metabolic state signals would be necessary to support the interpretation that IR56b functions as a primary state sensor.

(8) The manuscript suggests that IR20a and IR56b define two parallel and functionally independent pathways mediating nutrient detection and state-dependent preference, respectively. However, this conclusion is not fully supported by the current dataset. While the two receptors are shown to be expressed in distinct neuronal populations, the possibility of indirect interactions or convergence at downstream circuit nodes has not been excluded. Given that both pathways ultimately influence feeding behavior, it remains possible that they converge at higher-order interneurons or shared neuromodulatory circuits.

(9) In the state-dependent feeding assays (Figure 6), using H2O as a control introduces a severe masking effect. Salt-deprived flies actively suppress pure water intake to avoid osmotic shock, which artificially inflates the Preference Index (P.I.) for salt due to the denominator effect. To cleanly isolate salt preference from the thirst/osmotic drive, the authors will need to utilize an "isosmotic sucrose vs. isosmotic sucrose + salt" paradigm (Jaeger et al., 2018, eLife; Puri et al., 2026, PNAS).

Reviewer #3 (Public review):

Summary:

Drosophila, like other animals, use sophisticated taste systems with specialized chemoreceptors to identify gustatory cues in their environment. Multiple gustatory cues associated with a food source are often encountered simultaneously, but our understanding of how this sensory information is detected and integrated remains incompletely understood. This valuable study investigates how salt, amino acids, or their combination are detected by specific combinations of peripheral Ionotropic Receptors, leading to behavioral attraction. The authors show that distinct combinations of IR76b, IR25a, and IR20a confer sensitivity to salt, arginine, or both. They also show striking evidence that cells co-expressing IR25a/IR20a display a synergistic response to a mixture of sub-activating concentrations of these tastants. Together, these experiments lead to the conclusion that combinatorial expression of different subunits and synergistic responses to taste mixtures facilitates integration of taste cues beginning in the periphery. However, in its current form, key methodological details are missing or inadequately described, which complicates interpretation. Additionally, characterization is heavily focused on the population of IR20a+ neurons in the tarsi, while the response properties of the newly-identified, functionally distinct population in the labellum are investigated only through behavioral analysis, limiting the description of potentially additional IR20a complexes. Ultimately, more in-depth biochemical characterization of the IR complexes described will be required to fully support the conclusion that combinatorial assembly of distinct IR20a receptors enables peripheral integration of taste mixtures.

Strengths:

The authors characterize the expression pattern of IR20a in the tarsi as well as in the labellum, a tissue for which IR20a expression has been a point of debate. Multiple levels of analysis, including behavioral assays, physiological recordings, as well as ectopic and heterologous expression systems, are used to characterize the response properties of different combinations of IR subunits, demonstrating remarkably consistent behavior of the IR-complexes across cell types. Well-controlled genetic analysis and the use of multiple behavioral assays provide additional support for their results, including the surprising demonstration of synergistic responses to mixtures of tastants that supports the idea of peripheral integration of gustatory inputs. This report also identifies a distinct IR, IR56b, required for starvation-enhanced responses to salt.

Weaknesses:

(1) The title states that IR20a integrates L-arginine and salt signals via distinct subunit assemblies, though the paper lacks direct evidence that IR20a serves as a multimodal tuning receptor in distinct functional assemblies. Heterologous expression shows that co-expression of IR20a/IR25a confers sensitivity to Arg, IR76b confers sensitivity to NaCl, and IR20a/IR25a/IR76b co-expression confers sensitivity to both Arg and NaCl. This seems to be interpreted to mean that all three subunits are assembling into a single complex. However, current results do not show any difference in salt response when IR76b is expressed alone compared to alongside IR25a+IR20a. Without more direct evidence for co-assembly of all three subunits, it is equally plausible that the responses observed represent activity of distinct IR25a/IR20a and IR76b receptors for Arg and salt, respectively. In this model, genetic disruption resulting in expression of either IR25a or IR20a alone with IR76b could disrupt its activity or membrane trafficking (as seen here and in previous studies) while co-expression of both IR20a and IR25a relieves this inhibition by sequestering IR20a/IR25a into a distinct complex from IR76b. Direct biochemical characterization, for instance in the form of co-immunoprecipitation or FRET, will be required to differentiate between these possibilities.

(2) Key methodological details are missing throughout the manuscript. For instance, incomplete genotype and staining information is provided for images in Figure 1, making it difficult to interpret what is being shown. Additionally, for the calcium imaging methods, what is the imaging speed? How are max values calculated (is this the average of several images or just a single maximum)? How are ligands diluted and delivered to cells, and were they applied in a manner that allowed for subsequent washout?

(3) The composition of the S2 imaging bath buffer requires clarification. As described, the bath buffer appears to lack any Ca2+ or other IR-permeable cations. If this is indeed the case, more detail should be provided about why this bath buffer was selected and what this means for the source and mechanism of calcium responses observed, since it would not reflect direct IR-mediated transduction. It is also notable that addition of water gives such a detectable change in the tarsal preps.

(4) Visualization of IR20a driver activity in the labellum is interesting. Previous descriptions of labellar expression of IR20a range from no expression to expression in bitter neurons, so the current data linking IR20a to a different population of IR76b+ neurons warrants careful analysis in light of this discrepancy. However, some of the strongest presented evidence for expression is found in Figure 1, where the images are quite small, making it difficult to distinguish the morphology and sensillar innervation pattern of the cells labeled by the IR20a driver. In Figure 1A, several of the arrows do not appear to be associated with any visible fluorescence. It is similarly difficult to assess overlap. Including higher-resolution images and/or validating labellar expression, using antibodies, in situ hybridization, RT-PCR, or transcriptomics would strengthen these claims.

(5) Similarly, Figure 2 shows that IR20a is not required for Ca2+ responses to AAs or KCl in the legs, but is required for behavioral preferences and PER responses in the labellum. This suggests that IR20a receptors may function differently in different tissues, though direct evidence is lacking. Calcium imaging from a weakly expressed driver may be difficult, but electrophysiological recordings from relevant labellar sensilla or ectopic/heterologous reconstitution of the molecular receptors found there would give important insights into the response properties of these other IR20a receptor type(s) and could provide evidence for additional IR20a-containing complexes. The current paper focuses exclusively on IR25a/IR20a/IR76b, which do seem to reliably reproduce the Arg/NaCl responses observed in the tarsi, but even for the tarsal neurons it is unclear that this represents an exhaustive list of all the relevant IR20a-interacting subunits coexpressed in these cells. For instance, Koh et al., 2014 (PMID: 25123314) found several additional IR driver lines, including IR56b, were active in the 5v/s tarsal sensilla.

Author response:

We thank the editors and three reviewers for their careful evaluation and constructive feedback. We are pleased that our identification of IR20a as a multimodal tuning receptor required for both low-salt and arginine sensing in a distinct gustatory neuron population was recognized as a valuable contribution to sensory coding. We agree with the major points raised and outline our planned revisions below, organized thematically.

(1) Receptor assembly and integration model

Our genetic, heterologous, and calcium imaging data show functional cooperation among IR20a, IR25a, and IR76b but do not demonstrate physical association. In the revision we will replace terms like “distinct subunit assemblies” and “peripheral integration” with more cautious language such as “functional receptor combinations.” We will state explicitly that our data cannot resolve whether the three IRs form a single heteromeric complex, and we will discuss the alternative possibility that IR76b and the IR20a/IR25a pair function as separate receptors within the same neuron. This interpretation better accounts for the response patterns observed upon co-expression. Throughout the text and in a revised summary figure, we will clearly differentiate elements directly supported by data from those that remain inferential.

(2) Tarsal calcium imaging versus behavior

The mismatch between tarsal calcium imaging and behavioral arginine responses will be addressed directly. We will explain that tarsal recordings sample only a small subset of IR20a neurons, whereas proboscis extension and feeding assays predominantly engage the more numerous labellar sensilla, whose neurons may carry different receptor compositions. We will generate labellar imaging where feasible; if additional functional data cannot be obtained, we will acknowledge this limitation rather than overinterpreting the tarsal results.

(3) Synergy versus additivity

We will discuss behavioral and cellular data separately. Recognizing that true synergy is difficult to demonstrate in feeding and proboscis extension assays, we will adopt conservative terminology when interpreting those experiments. For cellular data, where mechanistic insight is stronger, we will present the evidence for functional synergy and discuss why the outcomes may differ between the cellular and organismal levels.

(4) Contextualizing prior IR76b and IR20a literatures

We will expand the Introduction and Discussion to cite more fully the works establishing IR76b as a low-salt sensor and IR20a’s role in amino-acid sensing, including the earlier report that IR20a overexpression can inhibit IR76b-dependent salt responses. We will clarify how our single-cell imaging and loss-of-function data obtained in the native context refine models derived from ectopic expression. In its endogenous setting, IR20a marks neurons narrowly tuned to amino acids such as arginine, and IR20a is strictly required for low-salt detection. These findings contrast with the earlier view that IR20a functions broadly as an amino-acid sensor or as a salt-response blocker.

(5) State-dependent modulation and IR56b

Our data do not identify IR56b as the direct molecular sensor of internal state. We will reframe IR56b as a necessary component for state-dependent modulation of low-salt preference and retract any claim that it is the sensor itself. We will also clarify that IR20a and IR56b define genetically separable peripheral pathways that may converge on downstream circuits.

(6) Methodological and presentation issues

We will address the following points raised across reviews:

(1) Co-localization: Higher-resolution confocal images and co-localization analysis will be provided.

(2) Summary model figure: A new figure will illustrate the distinct functions of IRs in low-salt and amino-acid taste, clearly indicating which aspects are directly supported and which are inferential.

(3) Feeding-assay control: We will either include an isosmotic sucrose control to avoid the water confound or explicitly discuss this limitation and temper the interpretation of feeding-preference results.

(4) S2 cell quantification: Complete details on response criteria, responder fractions, and statistical reporting will be added.

(5) Figure and supplementary corrections: All noted errors in figure legends, scale bars, citations, and supplementary-file mismatches will be fixed.

We are confident these revisions will bring our mechanistic claims into close alignment with the evidence and substantially improve the manuscript. We again thank the editors and reviewers for their detailed and helpful comments.