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

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


Figure 1 -
Figure 1-figure supplement 2. Gene structure of Ir60b locus, generation of Ir60b 3 and behavioral defect of Ir60b 1 in high salt avoidance.(A) Schematic of the Ir60b locus and Ir60b 3 allele.The Ir60b coding exon is indicated by the blue rectangle.Ir60b 3 was generated by ends-out homologous recombination by removing 768 base pairs as indicated.The red box indicates the insertion of the mini-white gene.The arrowheads (a-e) indicate the primers used for the PCR analyses in (B) and (C).(B) Confirmation of the deletion in Ir60b 3 by PCR using primers a and b (primer a: 5'-TTGGTGTTTACTCGAAAACA-3', primer b: 5'-GCATTCAGAATGTATCTTAG-3').(C) Confirmation of the deletion in Ir60b 3 by PCR using primers c and d, and e and f (primer c: 5'-CGAACTGCATGCGCAACAGT-3', primer d: 5'-TTGCTGCCTCCGCGAATTAA-3', primer e: 5'-TGTACTACTCACATTGTTCA-3', primer f: 5'-GATTGTGAGCAGCAGCAGCA-3').(D) Binary food choice assay testing control and Ir60b 1 flies with 1 mM sucrose versus 5 mM sucrose and 300 mM NaCl.n=10.The pairwise comparison was conducted using a Student t-test.Means ± SEMs.**p < 0.01.

Figure 2 -figure supplement 1 .
Figure 2-figure supplement 1. Assaying action potentials induced by different labellar bristles in response to 300 mM salt using tip recordings.(A) Tip recordings were performed on all S-type, I-type and L-type sensilla from control flies.n=12-14.(B) Tip recordings conducted on S3, S7, and L3 sensilla from the indicated Ir25a 2 and Ir76b 1 mutants, as well as the mutants expressing wild-type UAS-Ir25a and UAS-Ir76b transgenes expressed under control of the Ir25a-GAL4 and the Ir76b-GAL4, respectively.n=16-28.Multiple sets of data were compared using single-factor ANOVA coupled with the Scheffe's post hoc test.Statistical significances compared to the control line are indicated by the black asterisks, while the red asterisks indicate significant rescue compared to the corresponding mutant.Means ± SEMs.**p < 0.01.

Figure 2 -
Figure2-figure supplement 2. Two-way solid-food choice assay to assess whether the Gr2a GAL4 mutant exhibits a deficit in avoidance of high salt.The flies were given a choice between 1 mM sucrose versus 5 mM sucrose plus 300 mM NaCl in alternating wells of microtiter dishes.n=8.The pairwise comparison was conducted using a Student t-test.Means ± SEMs.

Figure 3 -
Figure 3-figure supplement 1. DrosoX system and measurement of food intake using strychnine and coumarin.(A) The DrosoX system is composed of a cassette

Figure 3 -
Figure 3-figure supplement 2. Two-way solid-food choice assay and DrosoX binary capillary feeding assay using 100 mM sorbitol with or without 300 mM NaCl.(A) Twoway solid-food choice assay to assess whether the Ir25a 2 , Ir60b 3 , and Ir76b 1 mutants exhibit a deficit in high salt avoidance.The flies were given a choice between 100 mM sorbitol versus 100 mM sorbitol plus 300 mM NaCl in alternating wells of microtiter dishes.n=6.(B and C) DrosoX assays used to test the relative volumes consumed by control, Ir25a 2 , Ir60b 3 , and Ir76b 1 flies when presented with capillaries containing either 100 mM sorbitol (a) or 100 mM sorbitol plus 300 mM NaCl (b).n=12.(B) Volumes of each of two food options.(C) Ingestion indexes (I.I) to indicate the relative consumption of the two foods.I.I formula: [Ingestion volume(b) -Ingestion volume(a)]/ [Ingestion volume(b) + Ingestion volume(a)].Multiple sets of data were compared using single-factor ANOVA coupled with Scheffe's post hoc test.Statistical significance compared with the controls.Means ± SEMs.*p < 0.05.**p < 0.01.

Figure 4 -figure supplement 1 .
Figure 4-figure supplement 1. Testing whether ectopic expression of Ir60b confers responses to 300 mM NaCl, measurement of intake of sucrose plus 300 mM NaBr using the DrosoX assay, and Ca 2+ response of Ir60b GRNs.(A) Tip recordings in response to 300 mM NaCl were performed on I5 and I9 sensilla of the indicated flies.UAS-Ir60b was expressed in Class B GRNs under control of the Gr33a-GAL4.n=10.(B and C) Droso-X assays used to test the relative volumes consumed by Ir25a 2 , Ir60b 3 , and Ir76b 1 flies when presented with capillaries containing either 100 mM sucrose (a) or 100 mM sucrose plus 300 mM NaBr (b).n=12.(B) Volumes of each of two food options.(C) Ingestion indexes (I.I) to indicate the relative consumption of the two foods.I.I formula: [Ingestion volume(b) -Ingestion volume(a)]/ [Ingestion volume(b) + Ingestion volume(a)].(D) GCaMP6f responses of Ir60b GRNs to sucrose only, NaCl only, or a combination of sucrose and NaCl.n=10.Multiple sets of data were compared using single-factor ANOVA coupled with Scheffe's post hoc test.Statistical significance compared with the controls.Means ± SEMs.*p < 0.05.**p < 0.01.