A Na+-selective High-salt Taste Receptor Mediates State-Dependent Sodium Aversion

A Na+-selective High-salt Taste Receptor Mediates State-Dependent Sodium Aversion | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A Na + -selective High-salt Taste Receptor Mediates State-Dependent Sodium Aversion Guangnan Qu , Bo Wang , View ORCID Profile Yan Chen doi: https://doi.org/10.1101/2025.10.07.680871 Guangnan Qu 1 Yunnan Key Laboratory of Cell Metabolism and Diseases, Center for Life Sciences, School of Life Sciences, Yunnan University , Kunming, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bo Wang 1 Yunnan Key Laboratory of Cell Metabolism and Diseases, Center for Life Sciences, School of Life Sciences, Yunnan University , Kunming, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yan Chen 1 Yunnan Key Laboratory of Cell Metabolism and Diseases, Center for Life Sciences, School of Life Sciences, Yunnan University , Kunming, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yan Chen For correspondence: yanchen{at}ynu.edu.cn Abstract Full Text Info/History Metrics Preview PDF Abstract Animals balance sodium intake by seeking low concentrations to meet physiological needs while avoiding toxic excess. However, how internal state selectively regulates high-sodium aversion remains unknown. Here, we identify the first Na + -selective high-salt taste receptor and demonstrate its essential role in internal-state-dependent sodium avoidance. In Drosophila , the ionotropic receptor IR11a, together with co-receptor IR25a, functions in bitter gustatory receptor neurons (GRNs) to mediate aversion to high Na + . Loss of IR11a abolishes neuronal and behavioral responses to Na + and Li + , while sparing detection of K + , Ca 2+ , and bitter compounds. Heterologous expression confirms that IR11a and IR25a form a Na + -selective channel. Notably, sodium deprivation or satiety specifically modulates Na + sensitivity in IR11a + GRNs—without altering responses to K + or bitter stimuli—and this modulation requires the IR11a/IR25a complex. These findings reveal a dedicated, state-dependent Na + -sensing pathway embedded within aversive taste cells, providing a peripheral mechanism for adaptive sodium consumption based on physiological need. Introduction Sodium is essential for survival, yet its excessive consumption is harmful, thus animals must ensure sufficient intake while avoiding toxic excess to maintain sodium homeostasis [ 1 ]. This regulation is achieved through bidirectional salt taste mechanisms: the attractive low-salt pathway promotes intake at beneficial concentrations (200 mM) [ 2 ]. Such a valence switch represents a conserved strategy across species [ 3 – 7 ]. In mammals, low-sodium detection is mediated by the amiloride-sensitive epithelial sodium channel (ENaC), which is expressed in dedicated taste receptor cells (TRCs) and selectively conducts Na⁺ and Li⁺, but not K⁺ [ 8 , 9 ]. In contrast, avoidance of high salt concentrations is primarily driven by bitter- and sour-sensing TRCs through a amiloride-insensitive, cation-nonselective mechanism, with anions and osmotic pressure also contributing to the aversive response [ 10 – 13 ]. Candidate molecular components of this pathway include bitter taste receptors (T2Rs), the carbonic anhydrase Car4, the Cl⁻-permeable channel TMC4, and possibly the non-selective cation channel TRPV1 [ 10 , 11 , 13 ]. However, the roles of T2Rs, Car4, and TRPV1 remain controversial, as it is unclear whether they function directly as high-salt receptors, modulate downstream signaling, or act through indirect mechanisms [ 2 ]. Recent studies indicate that TMC4 can contribute to high-salt responses, particularly via the Cl⁻ anion [ 10 ], yet other work has shown that pharmacological inhibition of anion channels or transporters does not alter these responses [ 14 , 15 ]. Thus, the molecular mechanisms underlying high-salt taste and the selective regulation of sodium avoidance remain unresolved. The behavioral response to salt and the coding logic of salt taste in Drosophila are similar to those in mammals [ 2 , 4 , 6 ]. Flies likewise employ a selective low-salt taste pathway, which relies on a heteromeric receptor complex of IR25a, IR76b, and IR56b in a subset of sweet-sensing gustatory receptor neurons (GRNs) to detect low sodium concentrations [ 5 , 16 ]. In contrast, high-salt responses are mediated by two GRN classes: bitter GRNs and Ppk23-expressing glutamatergic ( Ppk23 Glut ) GRNs [ 17 ]. Specifically, within the Ppk23 Glut GRNs, IR7c combines with IR25a and IR76b to form a broadly tuned receptor complex that detects multiple monovalent salts, including Na⁺, K⁺, and Cs⁺ [ 18 ]. However, the molecular basis of salt detection in bitter GRNs, and whether sodium-selective pathways exist for high-salt taste, have remained elusive. Sodium appetite is dynamically modulated by internal state: deprivation enhances attraction to sodium while satiety strengthens avoidance [ 17 , 19 , 20 ]. It has been shown that attractive sodium sensitivity is peripherally modulated by internal cues [ 21 , 22 ]. However, aversive salt taste pathways remain stable peripherally with modulation occurring centrally via forebrain subfornical organ (SFO) Ptger3 + neurons, which non-selectively enhance tolerance to various aversive chemicals during sodium deprivation [ 19 ]. Similarly in flies, state-dependent modulation of high-salt aversion occurs downstream of broadly tuned, cation-nonselective Ppk23ᴳˡᵘᵗ GRNs [ 17 ] or pharyngeal labral sense organ (LSO) neurons [ 23 ]. These mechanisms generate generalized salt tolerance but do not explain how animals can recalibrate sodium avoidance specifically while preserving defense against other cations, such as K + , that frequently co-occur with Na + in natural diets. Here, we identify IR11a as the core of a dedicated Na + -selective aversive pathway in Drosophila . Expressed in a subset of bitter GRNs, IR11a mediates aversion to high Na+ and Li + but not K + or Ca 2+ . While IR11a-mediated Li + responses require both coreceptors IR25a and IR76b, Na + detection depends only on IR25a. Heterologous expression confirmed that IR11a/IR25a forms a Na+-selective receptor, with IR76b broadening sensitivity to Li + . Crucially, unlike the stable and cation-nonselective IR7c/IR25a/IR76b pathway, IR11a + GRNs exhibit sodium-specific, state-dependent modulation mediated by IR11a/IR25a complex itself. This receptor complex therefore provides a peripheral mechanism for tuning sodium aversion in line with internal needs, complementing generalist salt pathways. Results A behavioral screening identifies IR11a as essential for NaCl avoidance To identify receptors responsible for high-salt detection, we conducted an RNAi-based behavioral screen. Using the pan-neuronal driver nSyb-GAL4 combined with tubulin-GAL80 ts , we conditionally knocked down candidate genes in adult flies. Two-choice feeding assays revealed that control flies ( nSyb-GAL4, tubulin-GAL80 ts ) exhibited temperature-dependent avoidance of 300 mM NaCl (reduced at 21°C compared to 31°C) but consistently avoided 400 mM NaCl at both temperatures ( Fig. 1A ). We therefore selected 400 mM NaCl for the screening assay. Download figure Open in new tab Fig. 1. IR11a mediates selective avoidance of high NaCl and LiCl concentrations (See also Fig. S1 ). (A) Avoidance responses of control flies ( nSyb-GAL4/+; Tub-GAL80 ts /+ ) cultured at restrictive (21°C) or permissive (31°C) temperatures to varying NaCl concentrations. (B) Pan-neuronal knockdown ( nSyb-GAL4/+; Tub-GAL80 ts /+ > UAS-RNAi ) of IR25a , IR7c , IR11a , and IR10a reduce high sodium avoidance. (C) IR11a 4 mutant has a four-nucleotide deletion (TTCC at positions 173-176 of exon 2). (D) IR11a mutants exhibit decreased avoidance of 5 mM suc + 400 NaCl versus 1 mM suc. (E) IR11a mutants exhibit increased attraction to 50 mM NaCl while decreased avoidance of 250 mM NaCl versus agarose. (F) Flies exhibit a significantly higher feeding rate on sucrose-supplemented salts than on salts alone, except for NaCl. (G) IR11a mutants specifically reduced high LiCl avoidance. (H-I) Restored avoidance of 250 mM NaCl (H) and LiCl (I) when IR11a is rescued. Concentrations in millimolar (mM). Data represent mean ± SEM (n = 10-45). Mann-Whitney for (A-G) , *p<0.05, *p<0.01, ***p<0.001. Kruskal-Wallis with Dunn’s multiple comparisons test for (H and I) , different letters indicate significant difference with p<0.05. Genotypes and statistical details are provided in Data S1 . Download figure Open in new tab Fig. S1. Genetic screening for receptor genes mediating high-salt avoidance using two-choice feeding assays (Related to Fig. 1 ). (A-D) Avoidance of 400 mM NaCl following pan-neuronal knockdown ( nSyb-GAL4/+; Tub-GAL80 ts /+ > UAS-RNAi ) of target genes under restrictive (21°C) and permissive (31°C) conditions. Significant increases (blue stars) denote differences versus controls. Given that knockdown of high salt receptor gene expression is estimated to reduce avoidance, genes associated with increased avoidance were not considered as candidates in this screening. Concentrations in millimolar (mM). Data represent mean ± SEM (n = 10-33). Mann-Whitney test for (A-D) , *p<0.05, **p<0.01, ***p<0.001. Genotypes and statistical details are provided in Data S7 Given that ionotropic receptors (IRs), pickpocket (Ppk) family, gustatory receptors (GRs), and transient receptor potential channels (TRP) family have been implicated in Drosophila gustatory sensation [ 24 – 27 ], we selected 64 candidate genes from these families available from lab stocks and Tsinghua Stock Center for screening. Our results showed that, in addition to the previously identified co-receptor IR25a and high-salt tuning receptor IR7c [ 18 ], knockdown of IR11a strongly reduced high-salt avoidance, and knockdown of IR10a exhibited a slight but statistically significant reduction ( Fig. 1B ). Knockdown of other candidates did not reduce avoidance to 400 mM NaCl ( Fig. S1 ). Based on the strength of its phenotype, we selected IR11a for further investigation. IR11a is required for LiCl and NaCl avoidance Next, we generated an IR11a mutant ( Fig. 1C ) via CRISPR/Cas9 and performed two-choice feeding assays to validate IR11a ’s role in high-salt avoidance. Consistent with the RNAi screen, IR11a mutants displayed significantly reduced avoidance of 400 mM NaCl ( Fig.1D ). To exclude potential confounding effects of sucrose, we tested NaCl versus plain agarose, with lower sodium concentration. Under these conditions, IR11a mutants showed reduced aversion to 250 mM NaCl and a slight but significant increase in attraction to 50 mM NaCl ( Fig. 1E ). These results suggest that, in addition to diminished avoidance, sodium-attraction pathways may be compensatorily enhanced in IR11a mutants. We next tested whether IR11a is required for the detection of other aversive compounds. Because flies generally prefer tastant-sucrose mixtures over tastant alone, except in the case of NaCl ( Fig. 1F ), we used a tastant-sucrose mixture versus sucrose to assess aversion to other salts and bitter compounds. In these conditions, IR11a mutants selectively exhibited reduced avoidance of 250 mM LiCl, while responses to KCl, CaCl₂, quinine, and denatonium remained unaffected ( Fig. 1G ). Targeted rescue of IR11a expression using IR11a-GAL4 fully restored avoidance to 250 mM NaCl and LiCl ( Fig. 1H–I ), confirming the specificity of the phenotype. IR11a is expressed in bitter GRNs mediating high NaCl and LiCl avoidance To identify the anatomical sites where IR11a mediates NaCl and LiCl avoidance, we analyzed the expression pattern of IR11a using IR11a-GAL4 [ 28 ]. Immunostaining revealed that this GAL4 constant labeling of GRNs in labellar sensilla S4, S6, and S10, with additional labeling in S8 sensilla in 40% of preparations ( Fig. S2A ). In the legs, expression was restricted to foreleg tarsal f5s sensilla ( Fig. S2B ), and in the pharynx, two neurons in the ventral cibarial sense organ (VCSO) were labeled ( Fig. 2A-D ). No expression was detected in the brain, gut, or Malpighian tubules ( Fig. S2C-D ). Download figure Open in new tab Fig. S2. Expression analysis of IR11a-GAL4 (Related to Fig. 2 ). (A) IR11a-GAL4 labels S4, S6, and S10 sensilla consistently in the labellum, with additional expression in S8 sensilla in 40% of preparations. (B) IR11a-GAL4 is exclusively expressed in f5s sensillum of the forelegs. (C) Expression and projection of IR11a-GAL4 in brain and ventral nerve cord. (D) No expression of IR11a is observed in gut and Malpighian tubule. Genotype: IR11a-GAL4/UAS-mCD8-GFP . Download figure Open in new tab Fig. 2. IR11a is expressed in bitter GRNs mediating high NaCl and LiCl avoidance (See also Fig. S2 ). (A-D) Co-localization analyses of IR11a - GAL4 with GR66a-LexA (bitter GRNs), GR64f LexA (sweet GRNs), IR76b - QF (coreceptor), and IR25a (coreceptor) in labellum, legs, and ventral cibarial sense organ (VCSO). (E-H) Silencing of IR11a + GRNs reduces avoidance of high concentrations of NaCl (E) and LiCl (F) , but does not affect avoidance of bitter compounds quinine (G) and denatonium (H) . (I) Artificial activation of IR11a + GRNs via ectopic capsaicin receptor expression exhibits avoidance capsaicin. Concentrations in millimoles (mM). Data represent mean ± SEM (E-I, n = 13-39). Kruskal-Wallis with Dunn’s multiple comparisons test for (E-I) ; different letters indicate significant differences with p<0.05. Genotypes and statistical details are provided in Data S2 . To identify the neuronal identity of IR11a + cells, we performed co-expression analyses using GR66a-LexA (bitter GRNs) [ 29 ], GR64f LexA (sweet GRNs) [ 30 ], IR76b-QF [ 5 ], and anti-IR25a antibodies [ 31 ]. IR11a-GAL4 is exclusively expressed in GR66a-LexA GRNs in labellar and legs ( Fig. 2A ), although only one of the two IR11a + VCSO GRNs co-expressed GR66a-LexA ( Fig. 2A ). All IR11a + GRNs co-expressed IR25a but not IR76b-QF or GR64f-LexA ( Fig. 2B – D), indicating their identity as a subset of bitter GRNs. To determine the behavioral role of IR11a+ GRNs, we silenced or activated them by expressing Kir2.1[ 32 ] or capsaicin receptor (VR1) [ 33 ], respectively, using IR11a-GAL4 , and conducted two-choice feeding assays. Silencing IR11a + GRNs reduced avoidance of high Na + and Li + , but did not affect aversion to bitter compounds such as denatonium or quinine ( Fig. 2E-H ), phenocopying IR11a mutants ( Fig. 1E, G ). Conversely, activation of IR11a + GRNs by capsaicin induced robust avoidance of capsaicin-laced food ( Fig. 2I ), demonstrating that these neurons are sufficient to drive aversive behavior. Together, these findings demonstrate that IR11a is expressed in a distinct subset of bitter GRNs that are both necessary and sufficient for mediating aversive responses to high concentrations of Na + and Li + . IR11a + GRNs mediate high sodium salt response through IR11a and IR25a Having established that both the IR11a gene and IR11a + GRNs are necessary for high-sodium and lithium avoidance, we investigated the cation selectivity of these neurons and the specific role of IR11a in mediating these responses. Single-cell calcium imaging of tarsal IR11a + GRNs (labeled by GR33a GAL4 ) revealed that these neurons exhibited dose-dependent increases in response to moderate to high NaCl concentrations, reaching a plateau at 500 mM NaCl ( Fig. 3A-B ). Additionally, they responded to 250 mM of various salts, including LiCl, NaBr, and KCl, as well as to 50 mM CaCl 2 and 1 mM denatonium ( Fig. 3C ). The salt responses were cation-dependent, as no activity was observed in response to 250 mM NMDG-Cl ( Fig. 3C ). Despite this broad tuning, IR11a mutants showed a selective deficit in their responses to Na + and Li + salts, and the reduction of calcium response for Na + and Li + was fully rescued by the reintroduction of IR11a ( Fig. 3C ), consistent with the specific requirement for Na + and Li + avoidance in two-choice feeding assays ( Fig. 1 ). Intriguingly, axonal imaging in the subesophageal zone (SEZ) confirmed that labellar IR11a + GRNs exhibited NaCl response profiles similar to those in legs and that IR11a is selectively required for Na + detection; however, these neurons lacked sensitivity to LiCl ( Fig. S3A-C ). This suggests the absence of key component required for Li + sensing in this tissue. Download figure Open in new tab Fig. S3. IR11a selectively required for high Na + responses in labellum GRNs (Related to Fig. 3 ). (A-B) IR11a + labellum GRNs exhibit dosage dependent respond to high concentrations of NaCl. (C) IR11a + labellum GRNs exhibit selectively impaired responses to 250 mM in IR11a mutants. Black stars indicate responses differing from H 2 O in control flies (p < 0.05). Concentrations in millimoles (mM). Data present mean ± SEM (n = 8-16). Mann-Whitney test for (B, C) , *p<0.05, **p<0.01, ***p<0.001. Kruskal-Wallis with Dunn’s multiple comparisons test for (C) ; different letters indicate significant differences with p<0.05. Genotypes and statistical details are provided in Data S8 . Download figure Open in new tab Fig. 3. IR11a collaborates with IR25a to selectively detect high sodium salts (See also Fig. S3 ). (A-B) IR11a + GRNs showed dosage dependent increasing respond to moderate to high NaCl concentrations, reaching a plateau at 500 mM NaCl. (C-E) IR11a + tarsal GRNs ( GR33a GAL4 f5s) exhibit selectively impaired responses to sodium and lithium salts in IR11a (C) and IR25a (D) mutants, while reduced responses to sodium salt only in IR76b mutants (E) , and rescued responses observed in rescue genotypes. (F) GR66a-LexA and IR76b-GAL4 coexpress in tarsal 5s sensillum. (G) Amiloride selectively inhibits NaCl responses in IR11a + GRNs (labeled by GR33a GAL4 f5s). (H) IR7c + GRNs display amiloride-insensitive responses. Black stars indicate responses differing from H 2 O controls (p < 0.05). Concentrations in millimoles (mM). Data represent mean ± SEM (n = 8-24). Black asterisks indicate responses differing from H 2 O in control flies (p < 0.05), while red asterisks denote significant differences between the two designated groups. The Mann-Whitney test was used to compare responses of different tastants with H₂O in control flies in (B, C, G-H) , *p < 0.05, **p < 0.01, ***p < 0.001. Dunn’s multiple comparisons test was used for comparisons among control, mutant, and rescue groups in (C-E) . Different letters indicate significant differences (p < 0.05). Genotypes and statistical details are provided in Data S3 . We next sought to examine whether the co-receptors IR25a and IR76b are required for salt detection in IR11a + GRNs. Calcium imaging showed that IR25a mutants selectively reduced responses to LiCl and NaCl, but did not alter responses to KCl, CaCl 2 , or denatonium in IR11a + GRNs, and the reduced response to LiCl and NaCl were fully rescued by neuron-specific reintroduction of IR25a ( Fig. 3D ). Interestingly, IR76b mutants exhibited selectively reduced responses to LiCl, but not NaCl, KCl, CaCl 2 , or denatonium ( Fig. 3E ). This requirement for IR76b in LiCl detection was unexpected, as initial expression analysis indicated that IR11a-GAL4 did not overlap with IR76b-QF ( Fig. 2C ). To resolve this discrepancy, we reassessed the expression of IR76b in IR11a + GRNs using IR76b-GAL4 [ 31 ]. As direct co-localization with IR11a-GAL4 was not feasible, we used GR66a-LexA as a marker for the bitter GRNs where IR11a is expressed. Co-expression of IR76b-GAL4 and GR66a-LexA was observed in tarsal f5s sensilla, confirming that IR76b is present in IR11a + GRNs ( Fig. 3F ). These data indicate that IR11a and IR25a form a core receptor complex for high-concentration sodium detection, whereas IR76b acts as an accessory subunit rather than a coreceptor required specifically for lithium detection. The Na + selectivity of the IR11a/IR25a complex is reminiscent of the amiloride-sensitive epithelial sodium channel (ENaC) in mammals [ 8 ], prompting us to test for amiloride sensitivity in Drosophila . Amiloride application selectively reduced NaCl-evoked calcium responses in IR11a + GRNs but had no effect on Na + responses in IR7c + GRNs, nor did it affect their responses to LiCl or KCl ( Fig. 3G-H ). This pharmacological evidence further supports a model where IR11a/IR25a forms an amiloride-sensitive, Na + -selective channel. Heterologous expression confirms IR11a/IR25a as a high sodium receptor To confirm that IR11a and IR25a form a high Na+-selective receptor, we ectopically expressed IR11a in pheromone-sensing tarsal f5a GRNs using Ppk23-Gal4 [ 29 ]. These neurons endogenously express the co-receptor IR25a but lack IR76b expression ( Fig. 4A-B ). Previous studies have shown that GRNs in the f5a sensillum do not respond to sugars, bitter compounds, acids, or salts [ 29 , 34 ]. Consistent with this, we confirmed that Ppk23-GAL4 GRNs in the f5a sensillum were unresponsive to NaCl, LiCl, or KCl ( Fig. 4C ). However, misexpression of IR11a selectively conferred their sensitivity to 250 mM NaCl, without inducing responses to other salts ( Fig. 4C ). This induced NaCl response was abolished in an IR25a mutant background but remained intact in an IR76b mutant ( Fig. 4D-E ), demonstrating that IR11a and IR25a form an IR76b-independent sodium receptor complex. Download figure Open in new tab Fig. 4. Misexpression of IR11a in pheromone-sensing GRNs confers NaCl sensitivity. (A) IR76b-QF does not co-express with Ppk23-GAL4 in the f5a sensilla. (B) Anti-IR25a shows that IR25a co-expresses with Ppk23-GAL4 in the f5a sensilla. (C) IR11a -misexpression in pheromone sensing GRNs ( Ppk23-GAL4 f5a) selectively confers NaCl response. (D-E) IR25a, but not IR76b, is required for 250 mM NaCl responses in Ppk23-GAL4 f5a GRNs following IR11a-misexpression. Concentrations in millimoles (mM). Data represent mean ± SEM (n =8-16). Mann-Whitney test for (C-E) , *p<0.05, **p<0.01, ***p<0.001. Genotypes and statistical details are provided in Data S4 . To assess the sufficiency of IR11a and IR25a in forming a sodium receptor and to examine their intrinsic receptor function, we expressed these receptors in Drosophila S2 cells. Calcium imaging revealed no salt responses in cells expressing either IR11a or IR25a alone, but robust responses to 250 mM NaCl upon co-expression of both subunits ( Fig. 5A-E ). These responses were not elicited by equimolar LiCl or KCl ( Fig. 5A–E ), confirming that IR11a and IR25a form a Na + -selective receptor complex. Consistent with previous studies [ 5 ], IR76b expression alone conferred NaCl sensitivity in S2 cells ( Fig. 5A, F ). Intriguingly, co-expression of IR25a with IR76b abolished IR76b-mediated NaCl responses in all tested concentrations ( Fig. 5G ), while co-expression of IR11a with IR76b reduced IR76b-mediated 50 mM NaCl responses but maintain 150 mM and 250 mM NaCl responses ( Fig. 5H ). In contrast, triple expression of IR11a, IR25a, and IR76b enabled responsiveness to both NaCl and LiCl, but not KCl ( Fig. 5I ), suggesting that IR76b expands the ionic sensitivity of the IR11a/IR25a complex. AlphaFold structural modeling predicted the following binding affinity hierarchy: 2IR11a + 2IR25a > IR11a + 2IR25a + IR76b > 2IR25a + 2IR76b > 4IR76b ( Table S1 ). This gradient likely accounts for the observed functional shifts, with higher-affinity heterotetrameric complexes outcompeting lower-affinity IR76b homomers ( Fig. 5F-I ). Together, these in vivo and in vitro findings demonstrate that IR11a and IR25a form a core sodium-detection complex, with IR76b modulating lithium sensitivity and expand sodium sensitivity in specific contexts. View this table: View inline View popup Download powerpoint Table S1. AlphaFold structural modeling predicts protein-protein interactions for different IR combinations (Related to Fig. 5 ). AlphaFold structural predictions revealed the following binding affinity hierarchy: 2 IR11a + 2 IR25a > IR11a + 2 IR25a + IR76b > 2 IR25a + 2 IR76b > 4 IR76b. Download figure Open in new tab Fig. 5. IR11a and IR25a form a high sodium receptor in S2 cells (See also Table S1 ). (A) heat maps of calcium responses in S2 cells expressing different IRs. (B-E) Heterologous expression in S2 cells confirms that IR11a and IR25a form high sodium receptor. (F) Heterologous expression of IR76b alone in S2 cells confer Na⁺-selective calcium responses. (G) Co-expression of IR25a and IR76b abolishes IR76b-mediated Na⁺ responses. (H) Co-expression of IR11a and IR76b abolishes IR76b-mediated responses to 50 mM NaCl but maintain responses to 150 mM and 250 mM NaCl. (I) Triple expression of IR11a/IR25a/IR76b confers responses to 250 mM LiCl and NaCl, but not to KCl. Concentrations in millimoles (mM). Data represent mean ± SEM (n = 11-20). Mann-Whitney test for (B-I) , *p<0.05, **p<0.01, ***p<0.001. Genotypes and statistical details are provided in Data S5 . IR11a and IR25a mediate state-dependent sodium taste plasticity Similar to mammals, Drosophila dynamically adjust their sodium salt feeding preferences in response to internal physiological states [ 16 , 17 ]. The Na + -selective properties of the IR11a/IR25a complex prompted us to investigate its role in modulating sodium avoidance under different internal conditions. Two-choice feeding assays revealed that wild-type flies exhibited reduced avoidance of 250 mM NaCl when salt-deprived compared to salt-fed controls ( Fig. 6A-B ). This state-dependent modulation was abolished in poxn mutants, which lack functional taste bristles, and in IR25a mutants, suggesting that peripheral GRNs and salt taste receptor mediated by IR25a control this behavioral plasticity ( Fig. 6A-B ). Download figure Open in new tab Fig. 6. IR11a and IR25a maintain state-dependent plasticity of sodium sensitivity (See also Fig. S4 ). (A) Poxn mutants, which lack peripheral taste sensilla, fail to exhibit adaptive avoidance of 250 mM NaCl during salt fed. (B-C) IR25a mutants lack adaptive avoidance of 250 mM NaCl during salt fed, with rescue responses occurring when IR25a is reintroduced via IR25a-GAL4 or IR11a-GAL4 , but not IR7c-GAL4 . (D-E) IR11a + GRNs show state-modulated NaCl responses (D) , while IR7c + GRNs exhibit static tuning (E) . (F-G) Both IR25a (F) and IR11a (G) are required for state-dependent plasticity of Na⁺ sensitivity in IR11a + GRNs. (H) The feeding ratio on 250 mM NaCl decreases in salt-fed control and IR11a rescue flies but not in IR11a mutants. (I) MAFE assays show that salt-fed control and IR11a rescue flies exhibited reduced feeding duration on 250 mM NaCl, whereas salt-fed IR11a mutants did not show this reduction. (J) MAFE assays show that neither control nor IR11a mutants exhibited reduced feeding duration on 20 mM sucrose. (K) The working model proposes that IR11a/IR25a integrates physiological state to modulate Na⁺ aversion. Concentrations in millimoles (mM). Data represent mean ± SEM (n = 8-15 for calcium imaging, while n = 5-29 for two-choice feeding assays and MAFE assays). Mann-Whitney test for (A-J) , *p<0.05, **p<0.01, ***p<0.001. Genotypes and statistical details are provided in Data S6 . Download figure Open in new tab Fig. S4. IR expression levels in IR11a + GRNs show no modulation by internal state (Related to Fig. 6 ). (A) qPCR analysis demonstrates unchanged transcriptional levels of IR11a , IR25a , IR76b , and IR7c in salt-deprived versus salt-fed conditions. (B-E) Immunostaining with anti-IR25a antibodies reveals no IR25a expression changes between salt-deprived and salt-fed conditions. Data present mean ± SEM (n = 3-21). Mann-Whitney test for (A, D-E) . Genotypes and statistical details are provided in Data S9 . We next examined the contributions of two high-salt-responsive GRN classes—those expressing IR11a and those expressing IR7c—to sodium avoidance plasticity. Restoration of IR25a expression in IR11a + GRNs rescued the deprivation-induced reduction in sodium avoidance, whereas IR7c-GAL4 -driven IR25a expression had no effect ( Fig. 6C ). These results identify IR11a + GRNs as key mediators of state-dependent modulation. Calcium imaging further revealed that IR11a + GRNs exhibited reduced NaCl-evoked activity in salt-deprived flies, while responses to KCl and the bitter compound denatonium remained unchanged ( Fig. 6D ). In contrast, IR7c + GRNs maintained stable NaCl responses regardless of salt status ( Fig. 6E ). These findings indicate that the IR11a pathway generates a tunable aversive signal that is suppressed under sodium-deprived conditions, whereas the IR7c pathway conveys a constant aversive signal, likely to prevent excessive salt intake irrespective of internal state. To determine whether the IR11a/IR25a receptor complex itself undergoes modulation, we analyzed IR11a and IR25a mutants. In both cases, IR11a + GRNs failed to exhibit state-dependent changes in NaCl responsiveness and responded similarly under salt-deprived and salt-fed conditions ( Fig. 6F-G ). This loss of plasticity was not accompanied by changes in the transcriptional levels of IR11a (a slight but statistically insignificant increase), IR25a , IR76b, or IR7c ( Fig. S4A ), or protein levels of IR25a ( Fig. S4B-E ), indicating that post-transcriptional modifications of the IR11a/IR25a complex or modulation of downstream signaling pathways likely mediate the observed sensitivity shifts. To directly assess whether IR11a affect state-dependent salt intake, we employed the manual feeding (MAFE) assay [ 35 ]. Wild-type flies displayed decreased feeding rates and shorter feeding duration for NaCl but not sucrose under salt-fed conditions ( Fig. 6H-J ). In contrast, IR11a mutants exhibited similar feeding behavior under both conditions, indicating a loss of state-dependent modulation, and this phenotype was rescued by reintroducing IR11a ( Fig. 6H-J ). Together, these results establish the IR11a/IR25a complex as a critical component of high sodium taste pathway that integrates internal state to regulate sodium intake. Discussion How animals dynamically balance sodium attraction and aversion according to internal need remains a central question. Current model describes high-salt aversion as a broadly tuned, cation-nonselective process that protects against diverse harmful ions, with these pathways thought to be insensitive to internal state [ 6 , 9 , 11 ]. This model, however, cannot explain how sodium aversion is selectively suppressed during depletion—when intake is essential—while avoidance of other cations remains intact. Our findings resolve this paradox by identifying a first dedicated Na + -selective high salt receptor that is uniquely capable of adaptive tuning at the periphery. We demonstrate that IR11a and IR25a form a Na + -selective receptor complex within a subset of bitter GRNs in Drosophila . Although these neurons respond broadly to salts and bitter compounds, loss of either subunit abolished Na + responses while sparing detection of K + , Ca 2+ , and bitter tastants ( Fig. 3C-D ). Reconstitution in S2 cells confirmed that IR11a/IR25a is sufficient to form a Na + -specific channel, while IR76b expands the receptor’s dynamic range and confers Li + sensitivity (Fig. E and I). This modular organization illustrates how molecular subunits can confer both selective and broader ionic sensitivities within the same sensory neuron. Why might flies require a Na + -selective high-salt receptor? We show that Na + sensitivity in IR11a + GRNs is suppressed during deprivation and enhanced after satiety, a modulation requiring the IR11a/IR25a receptor complex itself ( Fig. 6 ). In contrast, the IR7c/IR25a/IR76b pathway in Ppk23 Glut GRNs provides stable, nonselective detection of monovalent cations, unaffected by internal state. Our study thus demonstrates a model where IR7c + GRNs mediate generalist aversion to diverse salts, while IR11a + GRNs deliver sodium-specific, adaptive tuning that aligns intake with physiological demand ( Fig. 6K ). As the primary high-salt detection mechanisms in external taste neurons of the labellum and legs, these two parallel pathways collaborate to ensure salt avoidance while modulating sodium-specific aversion based on internal needs. This organization is ecologically advantageous: although rotting fruit—the natural food source of Drosophila —is typically Na + -poor and K + -rich, local factors like fermentation, microbial activity, desiccation, or anthropogenic salting can elevate Na + to harmful levels in specific niches. Under these conditions, a Na + -specific aversion pathway would be strongly favored by natural selection. Thus, the state-dependent IR11a pathway complements the generalist IR7c pathway to secure both nutrient balance and survival across variable ecological contexts. These external pathways further coordinate with sodium attraction mechanisms in sweet GRNs [ 16 , 22 , 36 ] and internal taste pathways in the pharyngeal LSO [ 37 , 38 ], enabling flies to dynamically modulate sodium intake while avoiding ingestion of potentially toxic non-sodium salts. Our findings also challenge the prevailing view that high-salt detection sensitivity is stable in external taste cells [ 17 , 19 ]. In contrast to models in which modulation arises only from central circuits [ 19 ], external GRN output [ 17 ], or pharyngeal internal taste cells [ 23 ], we show that the IR11a/IR25a complex functions as a Na + -selective high salt taste receptor in external aversive taste cells whose sensitivity is directly tuned by internal sodium state. This establishes peripheral taste receptors themselves as substrates for adaptive modulation. More broadly, our work reveals that even within broadly tuned gustatory systems, molecular specialization can evolve to meet specific physiological demands. The coexistence of generalist and selective pathways may represent a conserved design principle of sensory coding, balancing robustness with flexibility in nutrient regulation. The Drosophila ionotropic receptor (IR) family evolved within ancestral protostomes and shares ancestry with the AMPA/kainate clade of vertebrate non-NMDA ionotropic glutamate receptors (iGluRs), but has no direct homologs in vertebrates [ 39 ]. Nevertheless, the presence of a Na + -selective aversive pathway in Drosophila —a species not typically exposed to sodium-rich environments—suggests that this mechanism reflects deep evolutionary pressures rather than lineage-specific innovation. Our findings therefore raise the possibility that vertebrate taste systems may also harbor sodium-specific aversive receptors that remain to be identified. Materials and methods Flies husbandry and strains Flies were reared on standard food at 25℃ and 70% humidity with 12 hr light/dark cycles. The standard food comprises 72.53 g of agar, 520 g of corn meal, 1100 g of malt extract, 275 g of yeast, 31.27 g of propionic acid, and 14.07 g of tegosept in 10L of water. REAGENT &RESOURCE TABLE provides the full list of source reagents for standard food and fly strains used in this study. Molecular biology and transgenic flies pUAST-IR11a Genomic DNA sequence was used to generate UAS-IR11a transgenic flies. DNA was extracted from w 1118 flies, and the genomic sequences of IR11a were PCR amplified and cloned into the pUAST vector (See REAGENT&RESOURCE TABLE for primers). The sequences were confirmed by sequencing before injection. IR11a- gRNA and poxn -gRNA The guide RNAs were designed from the website: https://www.e-crisp.org/E-CRISP/ [ 40 ]. Two gRNAs of each gene were chosen and directly synthesized into the pCFD6 vector (See REAGENT&RESOURCE TABLE for primers). All plasmids were injected into embryos of our control w 1118 line by Fungene Biotechnology Co., Ltd. to obtain the transgenic flies. Transgenic gRNA flies were crossed with flies that express CAS9 in germ cells to obtain candidate mutants [ 41 ]. The crossing scheme to generate the IR11a mutant is as: P: ☿☿ hsFLP1,y 1 w 1118 ; ; UAS-Cas9.P, GAL4::VP16-nos.UTR,CG6325 MVD1 X IR11a-gRNA ♂♂ F1: ☿☿ hsFLP1,y 1 w 1118 ; ; UAS-Cas9.P, GAL4::VP16-nos.UTR,CG6325 MVD1 /IR11a-gRNA X FM7a/Y ♂♂ F2: ☿☿ FM7a/swa X IR11a (candidate mutant)/Y;; UAS-Cas9.P, GAL4::VP16-nos.UTR,CG6325 MVD1 /+(single male) ♂ ☿☿ FM7a/swa X IR11a (candidate mutant)/Y;; IR11a-gRNA /+ (single male) ♂ The crossing scheme to generate the poxn mutant is as: P: ☿☿ hsFLP1,y 1 w 1118 ; ; UAS-Cas9.P, GAL4::VP16-nos.UTR,CG6325 MVD1 X Poxn-gRNA ♂♂ F1: ☿☿ hsFLP1,y 1 w 1118 ; Poxn-gRNA/+ ; UAS-Cas9.P, GAL4::VP16-nos.UTR,CG6325 MVD1 /+ X If/Cyo ♂♂ F2: ☿☿ If/Cyo X Poxn (candidate mutant)/Cyo; UAS-Cas9.P, GAL4::VP16-nos.UTR,CG6325 MVD1 /+(single male) ♂ ☿☿ If/Cyo X Poxn (candidate mutant)/ Cyo; +/+ (single male) ♂ Candidate mutants were sequenced. IR11a 4 was identified as having a four-nucleotide deletion (TTCC at positions 173-176 of exon 2), while Poxn 84 possessed a single-nucleotide deletion (C at position 18 of exon 4), both causing frameshift mutations. The mutant lines were backcrossed to w 1118 for six generations before calcium imaging and behavioral analyses. Behavioral Analysis Two-choice feeding assays : two-choice feeding assays were performed as described previously [ 42 ], with minor modifications. Flies aged 8-10 days were used for the assays. Newly eclosed flies were maintained on standard food except when specified otherwise. For salt-deprived and salt-fed flies, newly eclosed flies were maintained on standard food for 2-4 days, then transferred to either salt-deficient food containing 0.7% agar and 563 mM sucrose, or salt-excess food containing 0.7% agar, 563 mM sucrose, and 50 mM NaCl, for 5 days. The 563 mM sucrose was used because it provides equivalent energy to our standard food. Each vial contained around 30 flies, starved for 18 to 20 hours in vials containing wet filter paper before the assays. Petri dishes (60 mm x 60 mm) were prepared immediately before each assay. Each dish was filled with 0.5% agarose mixed with tastants (including or excluding sucrose as specified), with one half containing blue dye (brilliant blue FCF at 0.125 mg/ml) and the other half containing red dye (sulforhodamine B at 0.2 mg/ml). Blue and red dyes were interchanged to eliminate bias due to dye color. Given that a higher proportion of flies fed on a mixture of tastant and sucrose compared to the tastant alone except for NaCl, we used a tastant-sucrose mix versus sucrose alone to assess the feeding preference or avoidance for tastants other than NaCl. The flies were chilled on ice and subsequently transferred to the dishes, where they were allowed to feed for 2 hours in dark conditions before being frozen for counting. Abdominal colors of flies in each dish were recorded under a stereomicroscope to assess feeding preferences. The abdomen of fed flies could exhibit blue, red, purple, or no coloration (indicating no feeding). The preference index (PI) was calculated using the formula: PI = [(number of flies with color 1) - (number of flies with color 2)] / (total number of fed flies). Data sets were discarded if the number of unfed flies exceeded half of the total fly count. MAFE assay Manual feeding (MAFE) assays were performed as described previously [ 35 ], with minor modifications. Flies used for MAFE assays received the same treatment as in the two-choice feeding assays. Flies were anesthetized on ice and affixed dorsally to glass slides using double-sided tape, with 20-30 flies per slide. The slides were then placed in an incubator at 25°C with 70% relative humidity for 1 hour to allow recovery. During the MAFE assay, each fly was first presented with water until it ceased drinking, followed by 250 mM NaCl solution until it stopped consuming the solution. This procedure was repeated for 3-4 rounds. Subsequently, 20 mM sucrose was applied using the same protocol for 3-4 rounds. If a fly did not consume 250 mM NaCl but drank 20 mM sucrose, it was scored as not feeding on 250 mM NaCl. Two datasets were collected: the proportion of flies that fed on 250 mM NaCl, and for those that did feed, the feeding duration was calculated. Both datasets were used in the Fig. 6H-J . Screening candidates for the high salt receptor UAS-RNAi lines targeting candidate genes were crossed with the pan-neuronal driver nSyb-GAL4 , in combination with the temperature-sensitive GAL4 inhibitor, tubulin-GAL80 ts ( nSyb-GAL4, Tub-GAL80 ts ). Flies were raised and maintained at 21°C to allow GAL80 to inhibit GAL4 activity, then shifted to 31°C for two days to restrict GAL80 activity before undergoing two-choice feeding assays. For the control, a subset of flies was raised and maintained at 21°C and was not shifted to 31°C before the assays. All two-choice feeding assays were conducted at 25°C. The two-choice assays presented flies with a choice between 400 mM NaCl + 5 mM sucrose and 1 mM sucrose to screen for potential high-salt receptor candidates. Flies with the same genotypes treated at 31°C that showed significantly decreased avoidance of high salt compared to those at 21°C were considered to carry candidate genes encoding high salt receptors. Immunohistochemistry Fly dissection and immunostaining were conducted as described previously [ 43 ]. Flies aged 6-10 days were used for dissection. Flies were anesthetized with CO 2 until immobile, and the labellum and legs were excised with a razor blade. The legs were cut between the third and fourth segments of the tarsi. The preparations were placed in 4% paraformaldehyde immediately after dissection, and the collected preparations were briefly centrifuged at 1000 rpm for 30 seconds to ensure complete immersion of the tissues in the fixation solution. The preparations were fixed on ice for 1 hour, then washed three times with PBST (1x PBS + 0.3% Triton X-100) at room temperature for 15 minutes each. For antibody staining, preparations were incubated in PBST + 5% horse serum solution for 2 hours at room temperature. Following this, they were rinsed twice and subsequently incubated with primary antibodies overnight at 4°C. Afterward, the preparations were washed three times with PBST for 15 minutes each. Subsequently, the samples were incubated in PBST + 5% horse serum for 30 minutes at room temperature, rinsed twice again, and then incubated with secondary antibodies for 3 hours at room temperature. Secondary antibodies were removed and preparations were washed three times with PBST for 15 minutes each. For mounting, preparations were placed on slides with a drop of antifade mounting medium, then covered with a coverslip and sealed with nail polish. Antibodies were diluted in PBST. Primary antibodies used in this study: chicken anti-GFP (1:2000), rabbit anti-RFP (1:2000), mouse anti-HA (1:500), mouse anti-nc82 (1:5000), and rabbit anti-IR25a (1:200). The IR25a antibodies were produced utilizing the same synthetic peptides described in Benton et al [ 31 ] as the antigen: SKAALRPRFNQYPATFKPRF and DVAEANAERSNAADHPGKLVDGV. Secondary antibodies: goat anti-chicken Alexa 488 (1:2000), goat anti-rabbit Alexa 488 (1:200), goat anti-mouse Alexa 568 (1:2000), goat anti-rabbit Alexa 568 (1:2000), and goat anti-mouse Alexa 647 (1:2000). Images were taken using a Zeiss LSM 800 confocal microscope with a 20X or 40X objective. Calcium imaging UAS-GCaMP6m was expressed under the control of different GAL4 drivers as specified in the experiments. Female flies aged 7-10 days were used for calcium imaging. Calcium imaging in the SEZ (subesophageal zone) : calcium imaging in the SEZ was conducted as described by Marella et al [ 33 ]. Flies were anesthetized on ice. The back of the neck and thorax were fixed in a custom chamber using glue. The proboscis was extended and immobilized with a needle. Antennae were removed, and a small window was cut in the cuticle around the SEZ to expose the brain. The exposed SEZ region was bathed in AHL solution (108 mM NaCl, 8.2 mM MgCl 2 , 4 mM NaHCO 3 , 1 mM NaH 2 PO 4 , 2 mM CaCl 2 , 5 mM KCl, 5 mM HEPES, pH 7.5). The sample was examined under a Nikon Ni-U upright microscope with a 40X water immersion objective. For labellum stimulation, a tastant solution in an injector mounted on a micromanipulator was delivered to the labellum for 5 seconds. Fluorescence recording of GCaMP started 5 seconds before stimulation and continued for 10 seconds after stimulation. The labellum was rinsed with water at the end of each recording and allowed to recover for 1 minute before the next recording. Each preparation was stimulated with different tastants in ascending concentrations. Calcium imaging of single GRNs in the legs : calcium imaging in the legs was performed as described previously [ 44 ]. A piece of double-sided tape was affixed to a glass-bottom petri dish (35 x 35 mm). The legs were cut between the tibia and femur, with the cut end coated with grease and affixed to the tape, leaving the 4 th and 5 th tarsal segments hanging off the tape. A drop of melted agarose (50-60°C) was placed on the tape to secure the preparation. Imaging was conducted using a Nikon Ni-U upright microscope with a 40X water immersion objective. The fluorescence of the cell body was monitored, using the adjacent area as an internal control. Tastants were applied with a pipette, with recording starting 10 seconds before tastant application and lasting for 2-3 minutes. The fluorescence changes in SEZ and legs were calculated using the same formula: ΔF/F (%) = (F - F0) / F0, where F is the fluorescence value, and F0 is the mean baseline value (the average fluorescence of the 5 seconds before stimulation). The maximum ΔF/F (%) value was used for statistical analysis. S2 cell culture, transfection, and calcium imaging S2 cell experiments were adapted from Asefa et al with modifications [ 45 ] . Drosophila S2 cells were cultured in Sf-900 TM II SFM medium supplemented with 10% fetal bovine serum (FBS) and 0.5% penicillin/streptomycin at 25°C without CO 2 supplementation. Cells were passaged every 3 days at a 1:2 to 1:3 dilution ratio. The genomic DNA sequence of IR11a and the cDNA sequences of IR25a, IR76b, and GCaMP6m were cloned into the pAc5.1 vector; pAc5.1-GFP was provided by Dr. Guoqiang Zhang. Calcium imaging was performed using a Nikon Ni-U upright microscope equipped with a 10× objective. Immediately before imaging, the culture medium containing transfection reagent was replaced with an isotonic bath buffer (10 mM HEPES, 10 mM glucose, 250 mM NMDG-Cl; pH 7.4; 520 mOsm). Approximately 2 minutes after buffer exchange, the bath buffer was gently removed, leaving only a minimal volume sufficient to immerse the cells. Baseline fluorescence (F 0 ) was recorded for 10 seconds. Stimulation was then initiated by adding ligand solutions containing x mM salt (NaCl, KCl, or LiCl), 10 mM HEPES, 10 mM glucose, and y mM NMDG-Cl (pH 7.4); the salt concentration (x) and NMDG-Cl concentration (y) were adjusted to achieve 520 mOsm. Fluorescence (F) was monitored continuously for 120 seconds. Fluorescence changes were calculated as: ΔF/F (%) = [(F – F 0 ) / F 0 ] × 100. Peak ΔF/F responses, following background subtraction (using adjacent non-fluorescent regions of interest), were quantified for statistical analysis. Quantification of IR25a expression in IR11a + GRNs To quantify IR25a expression in IR11a + GRNs, we dissected the fifth tarsal segments of GR33a Gal4 > UAS-mCD8-RFP flies maintained under salt-deprived or salt-fed conditions. Tissues were fixed and incubated overnight at 4 °C with rabbit anti-IR25a (1:200) and Rat anti-mCD8 (1:2000), followed by goat anti-rabbit Alexa 488 and goat anti-rat Alexa 568 (1:2000). All samples were processed in parallel and imaged on a Zeiss LSM 800 confocal microscope with identical laser power and acquisition settings. Maximum-intensity projections were generated in Zeiss Zen, and mean fluorescence intensities were measured in the green (488 nm) and red (568 nm) channels. A signal-free region served as background. Relative IR25a signal was calculated as (F green – B green )/(F red – B red ) for each leg segment. Quantitative RT-PCR Quantitative RT-PCR was performed to determine ionotropic receptor (IR) gene expression under salt-deprived and salt-fed conditions. Labella were dissected from female flies in triplicate biological replicates. Total RNA was extracted with the Quick-RNA Microprep Kit (Beijing Tianmo Biotech), and 500 ng RNA was reverse-transcribed using HiScript II Q RT SuperMix (+gDNA wiper) (Vazyme). Reactions (15 µl) contained 2× M5 HiPer SYBR Premix EsTaq (7.5 µl), 1:10-diluted cDNA (4 µl), 10 µM gene-specific primers (0.3 µl each), ROX reference dye (0.3 µl), and nuclease-free water (2.6 µl). Cycling conditions were 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Relative expression was calculated by the 2 −ΔΔCT method [ 46 ] with rp49 as the endogenous control. Primer sequences are provided in REAGENT&RESOURCE TABLE. Quntification and statistical analysis Normal distribution was evaluated, and nonparametric tests were used for non-normal data. The Mann-Whitney test was used to compare two groups, while the Kruskal-Wallis test with Dunn’s multiple comparisons was used for multiple group comparisons. Detailed quantification and statistical analysis methods are listed in Data S1-S9. REAGENT&RESOURCE TABLE View this table: View inline View popup Funding This work was supported by grants from the National Natural Science Foundation of China ( https://www.nsfc.gov.cn/ , No. 32070997 to Y.C), Applied Basic Research Foundation of Yunnan Province ( https://kjgl.kjt.yn.gov.cn/egrantweb/ , No. 2019FY003018 to Y.C.), Research Startup Funding of Yunnan University ( https://www.ynu.edu.cn/ , to Y.C.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Authors contribution G.Q. performed the screen, molecular biology, immunostaining, leg calcium imaging, and two-choice feeding assays, B.W. performed SEZ calcium imaging and two-choice feeding assays, G.Q., B.W., and Y.C. analyzed data, Y.C. conceived and supervised the project, designed experiments, and wrote the paper. Declaration of interests The authors declare no competing interests. Acknowledgments We thank Drs. Hubert Amrein, Wei Zhang, Zhihua Liu, Shan Jin, Hui Xiao, Guoqiang Zhang, the Bloomington Drosophila Stock Center, the Tsinghua Fly Center, and VDRC for fly strains and plasmids, and the Animal Center of Yunnan University for antibodies. We thank Yan Pan for assistance with the MAFE assay and Drs. Wei Zhang, Zhihua Liu, Bin Qi, and Qingfeng Chen for critical comments on the manuscript. References 1. ↵ Collaborators GBDD . Health effects of dietary risks in 195 countries, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017 . 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Share A Na + -selective High-salt Taste Receptor Mediates State-Dependent Sodium Aversion Guangnan Qu , Bo Wang , Yan Chen bioRxiv 2025.10.07.680871; doi: https://doi.org/10.1101/2025.10.07.680871 Share This Article: Copy Citation Tools A Na + -selective High-salt Taste Receptor Mediates State-Dependent Sodium Aversion Guangnan Qu , Bo Wang , Yan Chen bioRxiv 2025.10.07.680871; doi: https://doi.org/10.1101/2025.10.07.680871 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42003) Biophysics (21470) Cancer Biology (18624) Cell Biology (25533) Clinical Trials (138) Developmental Biology (13390) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40432) Molecular Biology (17200) Neuroscience (88681) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4828) Physiology (7653) Plant Biology (15161) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9826) Zoology (2271)