Mechanoreceptor genes are essential for swallow control of Drosophila.

(A) Swallow patterns of liquid food. Filling and emptying process constitutes a cycle. (B-D) Swallowing behavior of Tmc, nompC and piezo mutant flies. Pump frequency represents the swallowing speed, ingestion rate indicates the efficiency of food intake, while volume per pump shows how much a fly ingests with one pump. N = 8 to 15 in each group. (E) Double mutans of Tmc and nompC show more server dysphagia. n = 9 to 13 in each group. In all analyses, one-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons was used, and statistical differences were represented as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data were represented as means ± SEM.

Mechanosensory neurons and mechanoreceptors are essential to swallow.

(A) Blocking synaptic transduction of Tmc positive neurons leads to a lower pump frequency, and the flies display difficulty in cibarium emptying (emptying time >0.3s). n = 9 to 27 in each group. (B-C) Inhibiting Tmc-GAL4 and nompC-QF double-labeled neurons by Kir2.1 results in a lower pump frequency and intake volume per minute. n = 7 to 12 in each group. (D) Arbitrary intensity of cibarium when flies of different genotype swallow. The ordinate value was graphed using the opposite value of the original arbitrary intensity, so the declining line represents emptying process (the same for figure 3B). (E) Pump frequency after knocking down the expression level of the mechanoreceptor genes. n = 6 to 23 in each group. (F) Projection pattern of md-C neurons in the brain and cibarium. About two pairs of mutil-dendritic neurons are situated along the pharynx. Genotype: Tmc-GAL4 ; UAS-FRT-mCherry / nompC-QF ; QUAS-FLP. Scale bar = 50 μm. (G) Somata of md-C neurons situated in the cibarium but not in the cibarium. Genotype: XUAS-RedStinger; Tmc-GAL4, UAS-FRT-GAL80-STOP-FRT/ +; nompC-QF, QUAS-FLP/+. Scale bar = 50 μm. In all analyses, one-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons was used, and statistical differences were represented as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data were represented as means ± SEM.

Activation of md-C neurons led to dysphagia.

(A) Optogenetic activation of md-C neurons with CsChrimson during food intake could induce accelerated swallowing, incomplete filling (the expansion range of cibarium decreased to about half the maximum) and difficulty in filling. n=27. (B) Arbitrary intensity of cibarium when CsChrimson was stimulated. (C) Swallowing behavior of flies with or without md-C neurons stimulated. n = 11 to 13 in each group. Pump frequency was calculated only when fly consistently pumped at a normal range (observed dye boundary in the cibarium is wider than 90% width of mouthpart). ATR, all trans retinal. In all analyses, two-tailed unpaired t-tests were used, and statistical differences were represented as follows: **P < 0.01, and ****P < 0.0001. Data were represented as means ± SEM.

Interaction between motor neurons and md-C neurons revealed working pattern of swallow.

(A) md-C neurons labelled by GFP antibody (green) and muscles labelled by phalloidin (magenta) showed that they are in close proximity around cibarium. Genotype: Tmc-GAL4/UAS-FRT-mCD8-GFP; nompC-QF/QUAS-FLP. Scale bar = 50 μm. (B-C) GRASP signals between md-C and MNs could be observed in the SEZ area. Genotype: Tmc-GAL4, UAS-FRT-GAL80-STOP-FRT/UAS-nSyb-spGFP1-10, lexAop-CD4-spGFP11; nompC-QF, QUAS-FLP/MN-LexA. Scale bar = 50 μm. (D-F and G-I) Activation of Tmc+ neurons via P2X2 increased motor neuron activity. Fluorescence changes (ΔF/F0) of GCaMP6s in motor neurons after adding either ATP solution or water. n = 4 to 10, ∗p < 0.05, one-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons was used, error bars indicate mean ± SEM. Scale bar = 50 μm. (J) Significant signal changes could be detected of the md-C neurons’ termini at fly’s SEZ area after feeding fly with 0.5M sucrose solution when GCaMP6s is expressed in md-C neurons. Scale bar = 50 μm. (K) are traces of fluorescence changes. n = 4 to 6, ∗p < 0.05, two-tailed unpaired t-test was used, error bars indicate mean ± SEM. (L) Working model for md-C-MN-CPG controlling swallow of Drosophila.

Mutation of mechanoreceptors leads to a lower pump frequency caused by longer emptying time.

(A-B) Filling time and emptying of the cibarium in flies of different genotypes. n = 6 to 14. (C) Ratio of filling time dividing emptying time indicates the proportion of two processes in one cycle. n = 6 to 14. (D) Tmc and piezo double mutation showed a similar phenotype with single mutation for either gene in swallowing behavior. n = 9 to 32. (E-H) Proportion of time with incomplete emptying during swallowing when flies were fed with methylcellulose solution. n = 4 to 11. Data were represented as means. In all analyses, one-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons except nonparametric test in D was used, and statistical differences were represented as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data were represented as means ± SEM

Expression pattern of Tmc, nompC, piezo, Tmc & piezo and nompC & piezo in the brain and cibarium.

Scale bar= 50 μm.

Regulation of md-C neurons requires the inhibitory inter-neurons.

(A) Knocking down the expression of GABAA-R in md-C neurons caused a lower pump frequency. n = 16 to 25, one-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons was used in B and C while nonparametric test was used in A, and statistical differences were represented as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data were represented as means ± SEM.

(B) Flies expressing ReaChR in md-C neurons display dysphagia when stimulated by light. n=18.

(C) Flies with labellum ablated still showed response to light stimulation of md-C, indicating that md-L neurons are not necessary for control of swallow rhythm. n=11.