C. elegans shuts down its digestion for survival when fed harmful food, specifically Staphylococcus saprophyticus (SS).

(A) Microscopic images showing worms fed with SS arrested at the L1 stage three days after hatching.

(B) Schematic model illustrating our hypothesis: C. elegans can sense or evaluate inedible food, such as SS, and subsequently shut down their digestion to arrest development as a protective survival strategy.

(C) Schematic drawing and quantitative data of the food dwelling/avoidance assay. Yellow circles indicate the food spot for OP50 or SS bacteria, respectively. The animals were scored at the indicated times after L1 worms were placed on the food spot. The red point indicates the position of each worm. Data are represented as mean

± SD. Bar = 1000 μm. ***p < 0.001; **p < 0.01 by Student’s t-test.

(D) Schematic drawing, microscopic images, and quantitative data of the food choice assay. L1 worms were placed at the center spot (origin). OP50 (yellow) and SS (blue) bacteria were placed on opposite sides of the plate. The red point indicates the position of each worm. The percentage of worms on each spot was calculated at the indicated times. Data are represented as mean ± SD. Bar = 1000 μm. ****p < 0.0001; **p < 0.01 by Student’s t-test.

(E-F) Schematic drawing and quantitative data of the lifespan of animals fed with SS or OP50. L1 worms were seeded onto OP50 and grown to the L4 stage. L4 worms were then moved to SS or OP50 food to measure lifespan. **p < 0.01 by log-rank test. All data are representative of at least three independent experiments.

C. elegans senses SS and shuts down digestion through NSY-1.

(A) Developmental phenotype of wild-type N2 and nsy-1(ag3) mutant worms fed with SS bacteria. Data are represented as mean ± SD. Bar = 200 μm.. ****p < 0.0001 by Student’s t-test.

(B) Schematic drawing, microscopic images, and quantitative data of the food dwelling/avoidance assay. Yellow circles indicate the food spot for SS bacteria. The animals were scored 4 hours after L1 worms were placed on the food spot. The blue circle indicates the edge of the bacterial lawn, and the red point indicates the position of each worm. Data are represented as mean ± SD. Bar = 1000 μm. **p < 0.01 by Student’s t-test.

(C) Schematic drawing, microscopic images, and quantitative data of the food choice assay. L1 nsy-1(ag3) worms were placed at the center spot (origin). Heat-killed OP50 (yellow) and SS (blue) bacteria were placed on opposite sides of the plate. The red point indicates the position of each worm. The percentage of worms on each spot was calculated at the indicated times. Data are represented as mean ± SD. Bar = 1000 μm. **p < 0.01; ***p < 0.001 by Student’s t-test.

(D) Survival curves of wild-type N2 and nsy-1(ag3) mutant worms fed with SS bacteria. L4 worms, previously fed OP50 bacteria, were transferred to SS food to measure lifespan. ****p < 0.0001 by log-rank test.

All data are representative of at least three independent experiments.

NSY-1 plays a critical role in AWC neurons to inhibit SS digestion.

(A) Microscopic image showing the expression pattern of nsy-1. The head of an adult transgenic animal carrying Pnsy-1::GFP and Podr-1::RFP shows colocalization of nsy-1 and odr-1. Bar = 20 μm.

(B) Developmental progression of nsy-1(ag3) mutant worms carrying Podr-1::nsy-1::gfp (AWC neuron-specific expression) grown on SS bacteria. Control animals are labeled with white stars, and animals carrying the transgenes (rescued animals) are labeled with yellow stars. Data are represented as mean ± SD. Bar = 200 μm. ****p < 0.0001 by Student’s t-test.

(C) Developmental progression of wild-type N2 and AWC neuron-specific knockout nsy-1 animals (AWC nsy-1 KO) grown on SS bacteria. Data are represented as mean ± SD. Bar = 500 μm. ***p < 0.001 by Student’s t-test.

(D) Microscopic images show str-2::GFP, a marker for AWC neuron states, in L1-staged wild-type and nsy-1(ky397) mutant worms grown on OP50 or SS bacteria for 6 hours. AWC neuron positions are highlighted with red and yellow arrows. Bar = 20 μm.

(E-F) Percentage of animals with different AWC neuron states. nsy-1 mutation promotes an 2AWCON state under SS feeding conditions (E), with approximately 50% of animals exhibiting 2AWCOFF neurons when feeding on SS (F). Data are represented as mean ± SD. ****p < 0.0001 by Student’s t-test.

(G) Developmental progression of wild-type N2, tir-1(qd4), and nsy-1(ag3) mutant worms grown on SS bacteria. Data are represented as mean ± SD. Bar = 200 μm. ****p < 0.0001 by Student’s t-test.

All data are representative of at least three independent experiments.

NSY-1 inhibits animals from digesting SS by inducing str-130.

(A) Schematic illustration showing that "X" genes rely on NSY-1 to shut down SS digestion. "X" genes induced by SS food are dependent on NSY-1, and their induction aids in shutting down SS digestion.

(B) Venn diagram showing the overlap of genes that respond to SS and rely on NSY-1. The number of genes is indicated in the diagram (also see Table S1).

(C) Transcriptome analysis showing str-130 mRNA expression, which relies on

NSY-1 in response to SS. Data are represented as mean ± SD. ****p < 0.0001; **p < 0.01; *p < 0.05 by Student’s t-test.

(D) Developmental progression of wild-type animals treated with control RNAi or str-130 RNAi grown on SS bacteria. Data are represented as mean ± SD. Bar = 200 μm. ****p < 0.0001 by Student’s t-test.

(E) Microscopic images and quantitative data of AWC neuron states in L1 animals treated with control RNAi or str-130 RNAi grown on SS bacteria. Data are represented as mean ± SD. Bar = 20 μm. ***p < 0.001 by Student’s t-test (1AWCON/1AWCOFF: control vs str-130 RNAi).

(F) Developmental progression of nsy-1(ag3) mutant worms carrying Pstr-130::str-130::mCherry grown on SS bacteria. Control animals are labeled with white stars, and animals carrying transgenes are labeled with yellow stars. Data are represented as mean ± SD. Bar = 400 μm. ***p < 0.001 by Student’s t-test.

(G) Microscopic images and quantitative data of AWC neuron states in L1 animals carrying Pstr-130::str-130::mCherry. Transgenic animals with overexpression of

str-130 (carrying Pord-1::GFP as a co-injection marker) show an increased 2AWCOFF state. Data are represented as mean ± SD. Bar = 20 μm. ****p < 0.001 by Student’s t-test (2AWCOFF: Control vs Transgene).

All data are representative of at least three independent experiments.

NSY-1 mutation activates animals to digest SS by inducing insulin signaling.

(A) Schematic illustration showing that NSY-1 inhibits the expression of "Y" genes, which promote SS digestion. Some genes induced by the nsy-1 mutation under SS feeding conditions could facilitate SS digestion in the nsy-1 mutant.

(B) Venn diagram showing the overlap of genes that respond to SS but are limited by NSY-1. A total of 308 candidate genes induced by the nsy-1 mutation under SS feeding conditions could potentially promote SS digestion.

(C) Transcriptome analysis showing that ins-23 expression is induced in animals with the nsy-1 mutation under SS feeding conditions. Data are represented as mean ± SD. **p < 0.01; n.s. not significant by Student’s t-test.

(D) Developmental progression of nsy-1(ag3) mutant animals treated with control

RNAi or ins-23 RNAi grown on SS bacteria. Data are represented as mean ± SD. Bar = 500 μm. ****p < 0.0001 by Student’s t-test.

(E) Developmental progression of nsy-1(ag3), daf-2(e1370), and nsy-1(ag3);daf-2(e1370) double mutant animals grown on SS bacteria. Data are represented as mean ± SD. Bar = 500 μm. ****p < 0.0001 by Student’s t-test.

All data are representative of at least three independent experiments.

NSY-1 mutation promotes animals to digest SS through inducing intestinal bcf-1.

(A) Microscopic images and quantitative data showing fluorescence of Pbcf-1::bcf-1::GFP in L1-staged wild-type (WT) and nsy-1(ag3) mutant animals fed with OP50 or SS bacteria for 6 hours. Data are represented as mean ± SD. Bar = 100 μm. ****p < 0.0001 by Student’s t-test.

(B) Microscopic images and quantitative data showing fluorescence of Pbcf-1::bcf-1::GFP in L1-staged wild-type and AWC nsy-1 KO mutant (AWC neuron-specific knockout nsy-1 animals) fed with SS bacteria for 6 hours. Data are represented as mean ± SD. Bar = 50 μm. ***p < 0.001 by Student’s t-test.

(C) Developmental progression of wild-type N2, nsy-1(ag3), bcf-1(ok2599), and nsy-1(ag3);bcf-1(ok2599) double mutant animals grown on SS bacteria. Data are represented as mean ± SD. Bar = 200 μm. ****p < 0.0001; n.s. not significant by Student’s t-test.

(D) Microscopic images and quantitative data showing fluorescence of Pbcf-1::bcf-1::GFP in nsy-1(ag3) mutant animals treated with control RNAi or ins-23 RNAi under normal RNAi feeding conditions. Data are represented as mean ± SD. Bar = 200 μm. ****p < 0.0001 by Student’s t-test.

All data are representative of at least three independent experiments.

A model reveals a neural-digestive mechanism for evaluating harmful food.

(A) AWC neuron-expressed NSY-1 detects Staphylococcus saprophyticus (SS) as harmful food and shuts down digestion by inducing the AWCOFF neural circuit and NSY-1-dependent STR-130. This mechanism protects the animals and helps them avoid harmful food.

(B) Mutations in NSY-1 lead to SS digestion by activating the insulin/IGF-1 signaling (IIS) pathway and BCF-1 expression, thereby reducing the animals’ ability to avoid harmful food and decreasing their protection.