1. Developmental Biology
  2. Neuroscience

Neuronal detection triggers systemic digestive shutdown in response to adverse food sources in Caenorhabditis elegans

  1. Yating Liu
  2. Guojing Tian
  3. Ziyi Wang
  4. Junkang Zheng
  5. Huimin Liu
  6. Sucheng Zhu
  7. Zhao Shan  Is a corresponding author
  8. Bin Qi  Is a corresponding author
  1. Southwest United Graduate School, Yunnan Key Laboratory of Cell Metabolism and Diseases, State Key Laboratory of Conservation and Utilization of Bio-resources in Yunnan, Center for Life Sciences, School of Life Sciences, Yunnan University, China
https://doi.org/10.7554/eLife.104028.3
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  1. Yating Liu
  2. Guojing Tian
  3. Ziyi Wang
  4. Junkang Zheng
  5. Huimin Liu
  6. Sucheng Zhu
  7. Zhao Shan
  8. Bin Qi
(2025)
Neuronal detection triggers systemic digestive shutdown in response to adverse food sources in Caenorhabditis elegans
eLife 14:RP104028.
https://doi.org/10.7554/eLife.104028.3
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eLife Assessment

This important study investigates how signals from the nervous system can influence the response to different food sources. To demonstrate the role of specific neuronal and intestinal regulators in sensing food quality and modulating digestion, the authors present evidence through a combination of genetic screening, RNA-seq analysis, and functional studies. These findings shed light on an adaptive strategy to integrate food perception with physiological responses, with a mix of solid and convincing evidence supporting the work.

https://doi.org/10.7554/eLife.104028.3.sa0

Abstract

The ability to sense and adapt to adverse food conditions is essential for survival across species, but the detailed mechanisms of neuron-digestive crosstalk in food sensing and adaptation remain poorly understood. Here, we identify a novel mechanism by which Caenorhabditis elegans detect unfavorable food sources through neurons and initiate a systemic response to shut down digestion, thus safeguarding against potential harm. Specifically, we demonstrate that NSY-1, expressed in AWC neurons, detects Staphylococcus saprophyticus (SS) as an unfavorable food source, prompting the animal to avoid and halt digestion of SS. Upon detection, the animals activate the AWCOFF neural circuit, leading to a systemic digestive shutdown, which is mediated by NSY-1-dependent STR-130. Additionally, NSY-1 mutation triggers the production of insulin peptides, including INS-23, which interact with the DAF-2 receptor to modulate SS digestion and affect the expression of intestinal BCF-1. These findings uncover a crucial survival strategy through neuron-digestive crosstalk, where the NSY-1 pathway in AWC neurons orchestrates food evaluation and initiates digestive shutdown to adapt effectively to harmful food sources.

eLife digest

Eating is essential for survival – but not all food is safe. Spoiled or toxic meals can cause illness, so animals must distinguish good food from harmful food. While the brain helps animals smell and taste, it is less clear how the nervous system communicates with the digestive system to prevent harm.

The tiny worm Caenorhabditis elegans (C. elegans) is a powerful model organism in biology because it has a simple nervous system and a transparent body. Living in soil and feeding on bacteria, the worm encounters both harmless and harmful species. One such bacterium, Staphylococcus saprophyticus, is toxic to C. elegans. Previous work showed that worms can avoid poor-quality food, but the mechanisms behind this behavior were unknown.

Liu et al. investigated how C. elegans detects and responds to dangerous food by exposing the worms to S. Saprophyticus for one to four days and by using a combination of genetic and imaging approaches to study the activity of neurons. With this approach, the team identified a pair of neurons in the worm’s head, called AWC neurons, as key “taste sentinels.”

A protein located in these neurons, NSY-1, enabled the worms to recognize S. saprophyticus as a threat. This detection triggered a neural circuit (the AWCOFF state), sending a body-wide signal that shut down the digestive system. Without this protective mechanism governed by the nsy-1 gene, worms continued to digest the toxic bacteria and had a shortened lifespan.

Further experiments revealed that these neural signals also regulated hormone-like peptides and gut-specific genes, fine-tuning digestive activity. Thus, NSY-1 functions as a molecular sensor that links the nervous system to the gut, forming a direct communication line that helps the animal avoid harm.

These findings reveal a fundamental survival mechanism that may represent an ancient system shared across animals, including humans. Understanding this brain–gut crosstalk in worms could provide insights into how the human nervous system defends against foodborne pathogens and toxins and may also illuminate the biological basis of some digestive disorders.

However, further research is needed to determine whether similar signaling pathways exist in mammals. Identifying equivalent molecules in humans could open new avenues for understanding and treating digestive disorders and food-related illnesses.

Introduction

Food is a source of essential nutrients and also poses a risk of lethal toxins and pathogens. Animals, including humans, must respond to various food sources to ensure survival. The ability to detect and adapt to adverse food conditions is crucial for the survival of many species. Various sensory mechanisms have evolved to monitor food quality by detecting beneficial and harmful substances, including olfactory (Fiala, 2007; McLachlan et al., 2022; Sengupta et al., 1996), gustatory (Avery et al., 2021; Hukema et al., 2006; Scott, 2018), and gut chemosensory systems (Bargmann, 2006). Food allergies serve as a biological food quality control system, offering protection and benefits by promoting food avoidance behavior (Florsheim et al., 2021). Research by Plum et al., 2023 and Florsheim et al., 2023 provides evidence that the immune system’s allergic response communicates with the brain in mice, leading to food avoidance. This avoidance behavior acts as a defense strategy, reducing the risk of exposure to harmful substances, including allergens.

The digestive system functions by transporting food through the gastrointestinal (GI) tract, where it is broken down into molecules that can be absorbed and utilized by the body’s cells. Thus, shutdown of digestion may serve as a mechanism for eliminating indigestible or harmful substances, acting as a protective system in animals to avoid adverse food. Despite this, the interaction between neuronal food detection and intestinal digestion, particularly in assessing and adapting to harmful food, remains inadequately understood.

The free-living nematode Caenorhabditis elegans thrives in organic-rich environments where it encounters a variety of microorganisms as food (Félix and Braendle, 2010; Samuel et al., 2016; Schulenburg and Félix, 2017). C. elegans has evolved mechanisms to sense bacterial presence and food quality, which influence its feeding behaviors and digestive processes to adapt to its environment. Previous research has identified heat-killed Escherichia coli as low-quality food, which the nematode avoids using its food-quality evaluation systems, such as the FAD-ATP-TORC1-ELT-2 pathway (Qi et al., 2017) and the UPRER–immunity pathway (Liu et al., 2024). Additionally, we have found that certain bacteria, like Staphylococcus saprophyticus (SS), are classified as inedible, leading to shutting down digestion and stunted growth in the nematodes (Geng et al., 2022). However, we still need to determine whether SS represents harmful food for C. elegans and how the nematode senses SS and shuts down digestion to reject it. It is hypothesized that C. elegans may detect or assess inedible food, such as SS, and subsequently halt digestion as a survival strategy. This suggests that the cooperation between food sensing and digestive systems could form a systemic food quality control mechanism in animals, aimed at minimizing the adverse effects of harmful food.

In this study, we developed a model in C. elegans to investigate responses to harmful food and explored how the nematodes sense and avoid ingesting such food by shutting down their digestive systems. We identified a food quality control mechanism involving communication between neurons and the digestive system. This mechanism functions as a defense strategy to minimize the adverse effects of harmful food environments on the animals.

Results

Shutting down digestion as a protective mechanism for survival in larval C. elegans

Previous studies have shown that C. elegans cannot digest SS, which prevents their development (Figure 1A). In natural environments, C. elegans rely on various bacteria for nutrition and growth. However, SS is not a viable food source for C. elegans. Larval arrest, such as the dauer stage, serves as an adaptive mechanism for survival under unfavorable conditions, including limited food availability or extreme temperatures (Baugh, 2013; Baugh and Hu, 2020). We hypothesize that 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 (Figure 1B).

Figure 1 with 1 supplement see all
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Caenorhabditis 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 3 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. Scale 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. Scale 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.

Figure 1—source data 1

Numerical data of Figure 1C–F.

https://cdn.elifesciences.org/articles/104028/elife-104028-fig1-data1-v1.xlsx

Our previous research demonstrated that C. elegans can assess and avoid low-quality food, like heat-killed E. coli, to adapt to nutrient-deficient conditions (Liu et al., 2024; Qi et al., 2017). To determine if C. elegans also detect SS as an unfavorable food source, we conducted two behavioral assays: food dwelling/avoidance and food choice (Qi et al., 2017). In the food dwelling/avoidance assay, larval-stage animals exhibited strong discrimination against SS compared to the standard food, OP50 (Figure 1C). In the food choice assay, the animals preferred OP50 over low-quality food such as heat-killed E. coli (Figure 1—figure supplement 1A) or SS (Figure 1D). However, they could not distinguish between heat-killed E. coli and SS (Figure 1—figure supplement 1B). These results suggest that SS acts as an unfavorable food that C. elegans can detect and avoid.

In response to starvation, L1 larvae can enter a state of developmental arrest, pausing their growth to survive (Baugh and Sternberg, 2006). To test whether C. elegans shut down digestion of SS as a protective strategy upon sensing unfavorable food, we performed a survival assay on L1 larvae fed with SS. We found that larvae unable to digest SS still survived under SS feeding conditions (Figure 1—figure supplement 1C), similar to larvae under L1 starvation. This suggests that shutting down digestion may be a protective mechanism in larvae under SS feeding conditions.

Previously, we observed that activating larval digestion with heat-killed E. coli or E. coli cell wall peptidoglycan (PGN) enabled the digestion of SS as food (Hao et al., 2024). Additionally, when animals reached the L2 stage by feeding normal OP50 diet, they could utilize SS as a food source to support growth (Figure 1—figure supplement 1D). These findings suggest that once digestion is activated (via E. coli components or L2-stage maturation), worms gain the capacity to process SS as a viable food source, abolishing SS-induced growth impairment (Hao et al., 2024; Figure 1—figure supplement 1D).

If SS is indeed an unfavorable or toxic food for C. elegans, digesting it could result in physiological defects. We measured the lifespan of L4 stage animals fed with SS or OP50. We found that SS consumption shortened their lifespan (Figure 1E and F), indicating a cost associated with digesting unfavorable or toxic food.

In conclusion, our data suggest that larval-stage C. elegans can sense and evaluate SS as an unfavorable food source, leading to the shutdown of digestion to avoid consumption, thereby protecting them and allowing adaptation to an unfavorable food environment.

C. elegans sense SS and shut down digestion through NSY-1

We speculated that key factors in C. elegans are involved in sensing SS and shutting down its digestion. If these factors are mutated, the animals would fail to detect SS as an unfavorable food source and would utilize it (Figure 2—figure supplement 1A). To identify these factors, we conducted an unbiased forward genetic screen to find mutant animals that cannot sense SS, thereby allowing its digestion and supporting growth. One of the mutant alleles identified, ylf6, could digest SS and exhibited a growth phenotype under SS feeding conditions (Figure 2—figure supplement 1B). Whole-genome deep sequencing revealed that ylf6 carries two mutations in the nsy-1 gene (H929Y, Q1191 [stop codon*]) (Figure 2—figure supplement 1C).

To confirm that nsy-1 is essential for shutting down SS digestion, we used an independent nsy-1 mutant allele, ag3, and found that nsy-1(ag3) mutants also digested SS (Figure 2A). To determine whether nsy-1 is crucial for sensing SS, we performed two behavioral assays: food dwelling/avoidance and food choice. In the food dwelling/avoidance assay, larval stage nsy-1 mutant exhibited significantly impaired avoidance responses at both 4 h and 6 h but not at 8 h, suggesting that NSY-1 is essential for sustained aversion to SS food in the early response (Figure 2B). In contrast to wild-type N2 animals, nsy-1 mutants preferred SS when given a choice between two poor-quality foods, heat-killed E. coli and SS (Figure 2C, Figure 1—figure supplement 1B). These results suggest that nsy-1 is essential for C. elegans to sense and avoid SS.

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Caenorhabditis elegans senses Staphylococcus saprophyticus (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. Scale bar = 200 μm. ****p<0.0001 by Student’s t-test. n = number of animals which were scored. (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 at the indicated times 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. Scale bar = 1000 μm. *p<0.05; **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. Scale 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.

Figure 2—source data 1

Numerical data of Figure 2A–D.

https://cdn.elifesciences.org/articles/104028/elife-104028-fig2-data1-v1.xlsx

Next, we examined whether larvae that cannot sense SS and do not shut down digestion can adapt to SS environments. We measured the survival rate of nsy-1 mutants under SS feeding conditions and found that these mutants had a higher mortality rate (Figure 2D).

Overall, these results indicate that C. elegans sense and detect unfavorable food, such as SS, through nsy-1 and subsequently shut down digestion to protect themselves and enhance survival.

NSY-1 functions in AWC neurons to shut down SS digestion

The nsy-1 gene in C. elegans encodes a MAP kinase kinase kinase (MAPKKK) that operates in the AWC neurons (Kim et al., 2002; Sagasti et al., 2001), which are essential for chemotaxis and odor sensation. The primary role of nsy-1 in AWC neurons is to regulate the asymmetric expression of odorant receptors, contributing to neuronal asymmetry and diversity (Chuang et al., 2007; Sagasti et al., 2001). We hypothesized that the shutdown of SS digestion in C. elegans is mediated by nsy-1 function in AWC neurons.

Firstly, we constructed a Pnsy-1::GFP reporter strain and confirmed that nsy-1 is expressed in the AWC neurons (Figure 3A). Secondly, we expressed nsy-1 in the AWC neurons of nsy-1 mutant animals and observed that the transgenic animals rescued the indigestion phenotype (Figure 3B). This indicates that nsy-1 functions in AWC neurons to shut down SS digestion. Thirdly, we used CRISPR to construct a mutant strain that knocks out nsy-1 specifically in AWC neurons (Figure 3—figure supplement 1). We found that nsy-1 knockout in AWC neurons also resulted in the shutdown of SS digestion (Figure 3C). These results collectively suggest that NSY-1 is functional in AWC neurons and is crucial for shutting down SS digestion in C. elegans.

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NSY-1 plays a critical role in AWC neurons to inhibit Staphylococcus saprophyticus (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. Scale 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. Scale bar = 200 μm. ****p<0.0001 by Student’s t-test. n=number of animals which were scored. (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. Scale bar = 500 μm. ***p<0.001 by Student’s t-test. n=number of animals which were scored. (D) Microscopic images show Pstr-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 h. AWC neuron positions are highlighted with red and yellow arrows. Scale bar = 20 μm. (E, F) Percentage of animals with different AWC neuron states. nsy-1 mutation promotes a 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<0.01 by Student’s t-test. n=number of animals which were scored. (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. Scale bar = 200 μm. ****p<0.0001 by Student’s t-test. n=number of animals which were scored. All data are representative of at least three independent experiments.

Figure 3—source data 1

Numerical data of Figure 3B–G.

https://cdn.elifesciences.org/articles/104028/elife-104028-fig3-data1-v1.xlsx

Beyond its established role in AWC neurons, we detected NSY-1 expression in the intestine (Figure 3—figure supplement 2A). To assess intestinal NSY-1 function, we performed tissue-specific rescue experiments in nsy-1 mutants using the intestinal-specific vha-6 promoter. Intestinal expression of NSY-1 significantly suppressed the enhanced SS digestion phenotype in nsy-1 mutants (Figure 3—figure supplement 2B), demonstrating functional involvement of gut-localized NSY-1 in regulating digestive responses. We propose intestinal NSY-1 mediates this effect through innate immune signaling, consistent with its known pathway components. As previously established (Geng et al., 2022), the canonical PMK-1/p38 MAPK pathway functions downstream of NSY-1, with both sek-1 and pmk-1 knockdown enhancing SS digestion through immune modulation. This indicates intestinal NSY-1 suppresses digestion may act through PMK-1-mediated immune responses. Since neuronal NSY-1’s role in digestive control was previously undefined, we prioritized mechanistic analysis of its neuronal function in digestion regulation.

To determine whether NSY-1 in AWC neurons mediates SS sensory perception, we performed dwelling (avoidance) and food-choice assays using AWC-specific nsy-1 knockout and AWC-rescued strains (nsy-1(ag3); Podr-1::nsy-1). In dwelling assays, AWC-specific nsy-1 KO mutants exhibited significantly impaired SS avoidance at 6 h (Figure 3—figure supplement 3A), while AWC-rescued strains restored avoidance capacity at 2–6 h (Figure 3—figure supplement 3B). Food-choice assays further revealed that AWC nsy-1 KO mutants preferentially migrated toward SS (Figure 3—figure supplement 3C), whereas AWC-rescued showed no preference between SS and HK-E. coli (Figure 3—figure supplement 3D). These data conclusively demonstrate that NSY-1 acts in AWC neurons to mediate SS recognition and aversion behaviors.

AWC neurons exhibit OFF state in sensing SS food

In C. elegans, the expression of the str-2 gene in AWC neurons indicates the ON state (AWCON) (Sagasti et al., 2001), which is associated with high cGMP levels and lower calcium activity, enabling the neuron to respond to specific odors. Conversely, the absence of str-2 expression marks the OFF state (AWCOFF), characterized by different odor responses, low cGMP levels, and higher calcium activity (Troemel et al., 1999). We aimed to investigate (1) whether AWC neurons exhibit different states under normal food (E. coli OP50) versus unfavorable food (SS) conditions and (2) whether the state of AWC neurons affects the ability of C. elegans to digest SS.

Using str-2::GFP as a marker for AWC neuron states (Troemel et al., 1999), we found that wild-type animals feeding on normal OP50 food typically exhibit one AWCOFF and one AWCON neuron. However, when feeding on SS, the proportion of animals with the AWCOFF state increased, with approximately 50% of animals exhibiting two AWCOFF neurons (Figure 3D–F).

In nsy-1 mutant animals feeding on OP50, str-2::GFP is expressed in both AWC neurons (2AWCON), consistent with previous studies (Chuang and Bargmann, 2005; Chuang et al., 2007; Sagasti et al., 2001; Troemel et al., 1999; Figure 3D and E). Notably, both AWC neurons remained in the AWCON state in nsy-1 mutants feeding on SS (Figure 3D and E). These results suggest that SS feeding induces an AWCOFF state in wild-type animals, while the nsy-1 mutation promotes an AWCON state even under SS feeding conditions. This implies that the AWCOFF state may inhibit SS digestion, whereas the AWCON state promotes it.

The TIR-1-NSY-1-SEK-1-MAPK pathway plays a crucial role in regulating the asymmetric AWC cell fate decision. Previous studies have shown that str-2::GFP expression in both AWC cells (2AWCON phenotype) occurs in tir-1, nsy-1, and sek-1 mutant animals (Chuang and Bargmann, 2005; Sagasti et al., 2001; Troemel et al., 1999). We found that tir-1 mutant can grow under SS feeding conditions (Figure 3G), indicating that tir-1 mutant can digest SS similarly to nsy-1 mutants. This demonstrates that the AWCON state promotes SS digestion.

To confirm the importance of AWC state in SS digestion, we performed AWC-specific neuron ablation experiments using previously validated transgenic strain that expresses cleaved caspase under the AWC-specific promoter, ceh-36 (ceh-36p::caspase). Critically, worms with ablated AWC neurons completely failed to digest SS food (Figure 3—figure supplement 4), phenocopying the non-digesting state of wild-type worms on SS. This result directly confirms that functional AWC neurons are essential for initiating SS digestion, aligning with our model where the AWC-OFF state (induced by SS) inhibits digestion while the AWC-ON state promotes it.

Overall, our data suggest that the state of AWC neurons is critical for sensing food in C. elegans. When sensing SS food, animals exhibit an AWCOFF state, which shuts down digestion. Conversely, the AWCON state promotes the digestion of SS.

NSY-1 shuts down SS digestion through induction of STR-130

We have demonstrated that NSY-1 in AWC neurons detects unfavorable food and shuts down digestion (Figure 3). To investigate the genes regulated by NSY-1 in response to SS and their impact on SS digestion, we conducted a transcriptomic analysis on L1 larval animals fed with normal food (OP50) or unfavorable food (SS) for a short duration (4 h). We speculated that some genes induced by SS food are dependent on NSY-1 (Figure 4A), and their induction aids in shutting down SS digestion.

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NSY-1 inhibits animals from digesting Staphylococcus saprophyticus (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 Supplementary file 1). (C) Transcriptome analysis showing str-130 mRNA expression, which relies on NSY-1 in response to SS. Data are represented as mean ± SD. **p<0.01 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. Scale bar = 200 μm. ****p<0.0001 by Student’s t-test. n=number of animals which were scored. (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. Scale bar = 20 μm. ***p<0.001 by Student’s t-test (1AWCON/1AWCOFF: control vs str-130 RNAi). n=number of animals which were scored. (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. Scale bar = 400 μm. ***p<0.001 by Student’s t-test. n=number of animals which were scored. (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. Scale bar = 20 μm. ****p<0.001 by Student’s t-test (2AWCOFF: Control vs Transgene). n=number of animals which were scored. All data are representative of at least three independent experiments.

Figure 4—source data 1

Numerical data of Figure 4B–G.

https://cdn.elifesciences.org/articles/104028/elife-104028-fig4-data1-v1.xlsx

RNA-seq data analysis revealed 304 NSY-1-dependent candidate genes responding to SS (Figure 4B, Supplementary file 1). Enrichment analysis (Figure 4—figure supplement 1A, Supplementary file 2) of these candidate genes showed mainly associations with biotic stimulus, defense responses, xenobiotic stimulus, suggesting that NSY-1 positively regulates stress response pathways to protect animals under harmful food, SS, feeding conditions. Moreover, we found that sensory perception-related genes (sra-32, str-87, str-112, str-130, str-160, str-230) (Figure 4—figure supplement 1A, Supplementary file 2) were also enriched, with many genes being G protein-coupled receptors (GPCRs), which mediate odor sensing (Buck and Axel, 1991).

We further analyzed the dependence of these enriched GPCRs on NSY-1 under SS feeding conditions (Figure 4—figure supplement 1B) and found that str-130 is significantly upregulated in response to SS, with its function strongly being NSY-1 dependent (Figure 4C).

Using RNAi knockdown and the SS growth assay, we observed that RNAi of str-130, str-230, str-87, or str-112 significantly enhanced SS growth (Figure 4—figure supplement 2A), with str-130 RNAi exhibiting the most robust phenotype—phenocopying nsy-1(ag3) mutants. Crucially, none of these GPCR knockdowns further enhanced growth in nsy-1(ag3) mutants (Figure 4—figure supplement 2B), confirming their position downstream of NSY-1. These data establish str-130 as the dominant effector of NSY-1-mediated SS response regulation, while suggesting minor contributions from other GPCRs (str-230, str-87, str-112).

It has been shown that str-130 is expressed in AWCOFF neurons, based on transgenic GFP reporter strains, str-130p::GFP (Vidal et al., 2018). Our data also show that str-130 expression is induced in wild-type animals fed with SS (Figure 4C), where AWC neurons exhibit the AWCOFF state (Figure 3D and E). Therefore, it is possible that the high expression of str-130, regulated by nsy-1, alters the AWC state and inhibits SS digestion.

Firstly, we found that knockdown of str-130 in wild-type animals promoted SS digestion, thereby supporting animal growth (Figure 4D), and the proportion of animals with two AWCOFF neurons decreased (Figure 4E). Secondly, we found that overexpression of str-130 in nsy-1 mutant animals inhibited SS digestion, thereby slowing animal growth (Figure 4F), and the proportion of animals with two AWCOFF neurons increased (Figure 4G). These results demonstrate that NSY-1 promotes the AWCOFF state by inducing str-130 expression, which in turn inhibits SS digestion in C. elegans.

To definitively establish the epistatic relationship between NSY-1 and STR-130, we performed RNAi knockdown of str-130 in the nsy-1(ag3) mutant background and assessed development on SS food. We found that the str-130 RNAi did not further enhance the developmental capacity of nsy-1(ag3) mutant animals on SS (Figure 4—figure supplement 3). This epistasis confirms STR-130 functions strictly downstream of NSY-1 within the same genetic pathway. Together with our overexpression data (Figure 4F and G) showing neuronal str-130 rescue suppresses SS digestion in nsy-1 mutants, these results establish a linear signaling axis where NSY-1 primarily achieves functional inhibition of SS digestion through induction of the GPCR str-130.

NSY-1 mutation promotes SS digestion by inducing insulin signaling

NSY-1 mutation promotes the digestion of unfavorable food, such as SS, and supports C. elegans growth (Figure 2A). We hypothesize that, in addition to upregulating certain genes, such as str-130, to inhibit SS digestion, NSY-1 may also suppress certain genes to prevent nematodes from utilizing SS (Figure 5A). One possibility is that some genes, induced by the nsy-1 mutation under SS feeding conditions, could facilitate SS digestion in the nsy-1 mutant (Figure 5A).

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NSY-1 mutation activates animals to digest Staphylococcus saprophyticus (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. n=3 biological replicates. (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. Scale bar = 500 μm. ****p<0.0001 by Student’s t-test. n=number of animals which were scored. (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. Scale bar = 500 μm. ****p<0.0001 by Student’s t-test. n=number of animals which were scored. All data are representative of at least three independent experiments.

Figure 5—source data 1

Numerical data of Figure 5B–E.

https://cdn.elifesciences.org/articles/104028/elife-104028-fig5-data1-v1.xlsx

Our RNA-seq analysis identified 308+46 = 354 genes that are induced by the nsy-1 mutation under SS feeding conditions (Figure 5B, Supplementary file 3). However, among these 354 genes, 46 genes can also be induced in wild-type animals fed with SS, suggesting that these 46 genes may not be involved in digesting SS in nsy-1 mutants. Therefore, the 308 candidate genes induced by the nsy-1 mutation under SS feeding conditions could potentially promote animals to digest SS.

Enrichment analysis revealed that genes related to extracellular functions, such as insulin-related genes, are induced in nsy-1 mutant animals (Figure 5—figure supplement 1A, Supplementary file 4). Further analysis of insulin-related genes from the RNA-seq data showed that ins-23 is predominantly induced in nsy-1 mutant animals (Figure 5—figure supplement 1B), suggesting its potential role in promoting SS digestion.

To determine if insulin-like peptide genes were functionally responsible for the enhanced SS growth observed in nsy-1(ag3) mutants, we performed functional phenotypic screening using the SS growth assay (worm length assay). We individually knocked down each of these candidates (ins-22, ins-23, ins-24, ins-27) in the nsy-1(ag3) mutant background. Among these, only RNAi targeting ins-23 significantly suppressed the enhanced development of the nsy-1(ag3) mutant on SS (Figure 5—figure supplement 2, Figure 5D). This targeted functional screening revealed that ins-23 has the most robust and specific role in mediating the enhanced digestion phenotype downstream of NSY-1 loss, providing the critical justification for our subsequent focus on this particular insulin-like peptide.

Given that INS-23 is expressed in AWC neurons (Figure 5—figure supplement 3A, from CeNGEN), this suggests increased production and likely enhanced release of INS-23 from AWC neurons in the nsy-1(ag3) mutant background, which promotes SS digestion.

The insulin/insulin-like growth factor signaling (IIS) pathway, particularly through the DAF-2 receptor, integrates nutritional signals to regulate various behavioral and physiological responses related to food (Kodama et al., 2006; Ryu et al., 2018). It has been shown that INS-23 acts as an antagonist for the DAF-2 receptor to promote larval diapause (Matsunaga et al., 2018). To test whether ins-23 induction in nsy-1(ag3) mutants promotes SS digestion through its receptor, DAF-2, we constructed a nsy-1; daf-2 double mutant. We found that the SS digestion ability of the nsy-1 mutant was inhibited by the daf-2 mutation (Figure 5E). This suggests that the nsy-1 mutation induces the insulin peptide ins-23, which promotes SS digestion through its potential receptor, DAF-2.

To investigate whether DAF-2 acts as the gut-localized receptor for neuronal INS-23 signaling, we performed tissue-specific rescue experiments in the nsy-1(ag3);daf-2(e1370) double mutant. When DAF-2 was re-introduced specifically in the intestine (using the ges-1 promoter), we observed a significant suppression of SS digestion (Figure 5—figure supplement 3B), but not rescue digestive defect. This indicates that INS-23 induction in nsy-1 mutants promotes digestion independently of intestinal DAF-2 function.

As established in our prior work (Geng et al., 2022), SS exposure triggers phosphorylation of PMK-1 (P-PMK-1) in C. elegans, and pmk-1 mutants exhibit enhanced growth on SS. This confirms that PMK-1-mediated innate immune signaling actively regulates SS responsiveness and digestion. To address whether PMK-1 functions downstream of NSY-1 within our proposed model, we performed critical epistasis analyses. While we observed that nsy-1 mutation elevates ins-23 (indicating NSY-1 suppression of ins-23), knockdown of pmk-1 did not alter ins-23 expression levels (Figure 5—figure supplement 3C). This demonstrates that PMK-1 does not operate through the INS-23 pathway to regulate SS digestion. Thus, although both pathways respond to SS, the PMK-1-mediated innate immune response and the NSY-1/INS-23 axis constitute distinct regulatory mechanisms governing digestive adaptation.

NSY-1 mutation promotes SS digestion through regulation of intestinal BCF-1

In our previous study, we found that heat-killed E. coli promotes SS digestion in C. elegans (Geng et al., 2022), a process requiring intestinal BCF-1 (Hao et al., 2024). In the absence of BCF-1, the digestive capability of the animals is significantly reduced (Hao et al., 2024). This led us to investigate whether NSY-1 in AWC neurons regulates intestinal bcf-1 expression.

Firstly, we used a BCF-1::GFP reporter to measure bcf-1 expression in nsy-1 mutant animals. We found that mutation of nsy-1 induced bcf-1 expression in animals fed either SS or OP50 food (Figure 6A). Additionally, we confirmed that nsy-1 mutation in AWC neurons also induced intestinal bcf-1 expression under SS feeding conditions (Figure 6B). This data indicates that NSY-1 in AWC neurons inhibits intestinal bcf-1 expression, implying that nsy-1 mutation promotes SS digestion through BCF-1.

Figure 6 with 1 supplement see all
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NSY-1 mutation promotes animals to digest Staphylococcus saprophyticus (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 h. Data are represented as mean ± SD. Scale bar = 100 μm. ****p<0.0001 by Student’s t-test. n=number of animals which were scored. (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 h. Data are represented as mean ± SD. Scale bar = 50 μm. ****p<0.0001 by Student’s t-test. n=number of animals which were scored. (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. Scale bar = 200 μm. ****p<0.0001 by Student’s t-test. n=number of animals which were scored. (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. Scale bar = 200 μm. ****p<0.0001 by Student’s t-test. n=number of animals which were scored. All data are representative of at least three independent experiments.

Figure 6—source data 1

Numerical data of Figure 6A–D.

https://cdn.elifesciences.org/articles/104028/elife-104028-fig6-data1-v1.xlsx

Next, we constructed a nsy-1; bcf-1 double mutant and analyzed the growth of these animals on SS. We found that the digestive ability of nsy-1 mutants was inhibited by the mutation of bcf-1 (Figure 6C). Together, our data suggest that the increased digestion ability in nsy-1 mutant animals is dependent on the intestinal digestion factor BCF-1.

We then asked how nsy-1 regulates intestinal bcf-1 expression. Since nsy-1 mutation induces the insulin peptide ins-23, which promotes SS digestion, we tested whether the induction of intestinal bcf-1 by nsy-1 mutation is also mediated through INS-23. We found that the bcf-1::GFP level decreased in nsy-1 mutant animals following ins-23 RNAi treatment (Figure 6D). This suggests that nsy-1 mutation activates bcf-1 expression in the intestine, which requires INS-23.

To test whether INS-23 acts in AWC neurons to regulate intestinal BCF-1, we generated AWC-specific knockdown strains, which were achieved by rescuing sid-1 cDNA expression under the ceh-36 promoter in a sid-1(qt9);BCF-1::GFP background.

We found that AWC-restricted ins-23 knockdown significantly reduced intestinal BCF-1::GFP expression (Figure 6—figure supplement 1A). This confirms that INS-23 functions within AWC sensory neurons to activate intestinal BCF-1, consistent with NSY-1’s upstream inhibition of INS-23 in this neuronal subtype.

NSY-1 promotes the AWCOFF state through STR-130 to suppress SS digestion. To determine whether AWC-expressed STR-130 regulates intestinal BCF-1 expression, we observed that AWC neuron-specific RNAi of str-130 elevated intestinal BCF-1::GFP expression (Figure 6—figure supplement 1B). This demonstrates that STR-130 functions in AWC neurons to repress BCF-1 expression in the intestine.

Discussion

This study in C. elegans reveals a neural-digestive mechanism for evaluating harmful food (Figure 7). The neuron-expressed NSY-1 protein detects SS as unsafe food, triggering a digestive shutdown via the AWCOFF neural circuit and the NSY-1-dependent STR-130. Mutations in NSY-1 lead to SS digestion, activating the insulin/IGF-1 signaling (IIS) pathway and BCF-1 expression. These findings highlight a food quality evaluation strategy where neurons communicate with the digestive system to assess food safety, providing insights into how animals adapt to toxic food environments.

Figure 7
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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 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.

The identification of NSY-1 in AWC neurons as a key player in detecting SS and initiating digestive shutdown is particularly intriguing. NSY-1, a MAP kinase (MAPKKK), is known to regulate the asymmetric expression of odorant receptors, contributing to neuronal asymmetry and diversity (Chuang and Bargmann, 2005; Chuang et al., 2007; Sagasti et al., 2001; Troemel et al., 1999). Previous research has demonstrated that AWC neurons are pivotal in chemotaxis and sensory processing (Bargmann et al., 1993; Troemel et al., 1997; Wes and Bargmann, 2001). Our findings extend its function to include food quality assessment, showing that NSY-1 can trigger a digestive shutdown via the AWCOFF neural circuit.

This neural circuit appears to be a crucial component of a systemic food quality control mechanism, allowing animals to adapt effectively to harmful food sources. The state of AWC neurons (AWCON or AWCOFF) directly influences the animal’s ability to digest SS, with the AWCOFF state inhibiting digestion and the AWCON state promoting it. The finding that activation of the AWCOFF neural circuit leads to a systemic digestive shutdown mediated by NSY-1-dependent GPCR(STR-130) is another notable advancement. GPCRs are well-known for their roles in sensory perception (Julius and Nathans, 2012; Troemel et al., 1995). Our results suggested that GPCR STR-130 plays a role in shutting down digestive processes while maintaining AWCOFF states for evaluating harmful food.

Our study underscores the critical role of insulin signaling pathways in mediating the effects of neuronal detection on intestinal functions. Upon detection of SS by AWC neurons, NSY-1 inhibits the expression of insulin-like peptides, particularly INS-23. These neuronal peptides promote the expression of BCF-1, a key regulatory factor in digestion (Hao et al., 2024). Once ins-23 was inhibited by neuronal NSY-1, the intestinal BCF-1 level is also reduced, which in turn shut down digestion. We found that the digestive ability of nsy-1 mutants was totally inhibited by the mutation of bcf-1 (Figure 6C), suggesting that the increased digestion ability in nsy-1 mutant animals is mainly dependent on the intestinal digestion factor BCF-1. This regulatory cascade highlights the intricate link between neuronal signals and gut responses, ensuring an adaptive reaction to harmful food. We speculated that except for INS-23 there should be other factors as signaling regulated by neuronal NSY-1 to inhibit intestinal digestion factor BCF-1 for digestion shutdown.

Future studies should focus on delineating the precise molecular pathways linking NSY-1 signaling in AWC neurons to digestion regulation in the gut. Identifying the role of other sensory neurons in food quality assessment and their interactions with the digestive system may uncover new facets of neuron-gut communication and adaptive responses.

In summary, our study reveals a sophisticated mechanism in C. elegans that integrates neuronal detection of harmful food sources with systemic digestive responses. This neuron-digestive crosstalk is crucial for maintaining organismal homeostasis and survival in the presence of toxic food sources. The findings suggest that similar pathways may exist in other species, including humans, providing a foundation for future research on food safety, toxin avoidance, and the neural regulation of digestive processes. This has important implications for public health, as it illuminates the biological mechanisms underlying foodborne illness and the body’s defenses against such threats.

Materials and methods

C. elegans strains and maintenance

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Nematode stocks were maintained on nematode growth medium (NGM) plates seeded with bacteria (E. coli OP50) at 20°C.

The following strains/alleles were obtained from the Caenorhabditis Genetics Center (CGC) or as indicated:

  1. The following strains were obtained from CGC:

    • N2 Bristol (wild-type control strain);

    • AU3: nsy-1(ag3);

    • ZD101: tir-1(qd4);

    • RB1971: bcf-1(ok2599);

    • KU25: pmk-1(km25);

    • KU4: sek-1(km4);

    • PY7502: oyIs85[ceh-36p::TU#813+ceh-36p::TU#814+srtx-1p::GFP+unc-122p::DsRed]

    • CB1370: daf-2(e1370); shared from Mintie Pu lab

    • CX3695: str-2::gfp+lin-15(+); shared from Huanhu Zhu lab

    • CX4998: str-2::gfp+lin-15(+);nsy-1(ky397); shared from Hongyun Tang lab

  2. The following strains were obtained from published papers:

  3. The following strains were constructed by this study:

    • YNU186: ylfEx124[Pnsy-1::gfp;Podr-1::rfp] was constructed by injecting

    • plasmid Pnsy-1::nsy-1::gfp with Podr-1::rfp in N2 background;

    • YNU189: Pbcf-1::bcf-1::gfp::3xflag;nsy-1(ag3) was constructed by crossing PHX4067[Pbcf-1::bcf-1::gfp::3xflag] with AU3[nsy-1(ag3)];

    • YNU238: ylf6(nsy-1, EMS mutant);

    • YNU465: ylfEx252[Podr-1::nsy-1::gfp;Podr-1::rfp;nsy-1(ag3)] was constructed by injecting plasmid Podr-1::nsy-1::gfp with Podr-1::rfp in nsy-1(ag3) background;

    • YNU466: ylfEx253[Pvha-6::nsy-1::gfp;Podr-1::rfp;nsy-1(ag3)] was constructed by injecting plasmid Pvha-6::nsy-1::gfp with Podr-1::rfp in nsy-1(ag3) background;

    • YNU488: ylfEx259[Pstr-130::str-130::mcherry;Podr-1::rfp;nsy-1(ag3)] was constructed by injecting plasmid Pstr-130::str-130::mcherry with Podr-1::rfp in nsy-1(ag3) background;

    • YNU491: ylf57, AWC neuron specific knock out nsy-1 strain was constructed by injecting plasmid pDD162[Podr-1::Cas9+Pu6::nsy-1-sg], nsy-1 repair template (synthesis from Tsingke), pDD162[Peft-3::Cas9+Pu6::dpy-10-sg], dpy-10 repair template(synthesis from Tsingke) in PHX4067(Pbcf-1::bcf-1::gfp::3xflag) background;

    • YNU501: nsy-1(ag3);bcf-1(ok2599) double mutant was constructed by crossing RB1971[bcf-1(ok2599)] with AU3[nsy-1(ag3)];

    • YNU508: ylfEx266[Pstr130::str-130::mcherry;Podr-1::rfp;nsy-1(ag3)] was constructed by injecting plasmid Pstr130::str-130::mcherry with Podr-1::rfp in str-2::gfp+lin-15(+) background;

    • YNU517: nsy-1(ag3);daf-2(e1370) double mutant was constructed by crossing CB1370[daf-2(e1370)] with AU3[nsy-1(ag3)];

    • YNU729: ylfEx351[Pges-1::daf-2::gfp;Podr-1::rfp;nsy-1(ag3);daf-2(e1370)] was constructed by injecting plasmid Pges-1::daf-2::gfp with Podr-1::rfp in nsy-1(ag3);daf-2(e1370) background;

    • YNU732: ylfIs48[Podr-1::nsy-1::gfp;Podr-1::rfp;nsy-1(ag3)] was constructed by injecting plasmid Podr-1::nsy-1::gfp with Podr-1::rfp in nsy-1(ag3) background;

    • YNU730: ylfEx352[Pceh-36::sid-1::mcherry;pRF4(rol-6);Pbcf-1::bcf-1::gfp::3xflag;sid-1(qt9)] was constructed by injecting plasmid Pceh-36::sid-1::mcherry with pRF4(rol-6) in Pbcf-1::bcf-1::gfp::3xflag;sid-1(qt9) background;

    • YNU731: ylfEx353[Pins-23::ins-23::gfp;Podr-1::rfp] was constructed by injecting plasmid Pins-23::ins-23::gfp with Podr-1::rfp in N2 background.

Bacteria strains

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E. coli-OP50 (from CGC) and SS (from ATCC) were cultured at 37℃ in LB medium. A standard overnight cultured bacteria was then spread onto each Nematode growth media (NGM) plate.

Generation of transgenic strains

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  1. To construct the C. elegans plasmid for expression of nsy-1, 1527 bp promoter of nsy-1 was inserted into the pPD95_77-gfp vector. DNA plasmid mixture containing Pnsy-1::GFP (20 ng/ul) and Podr-1p::RFP(50 ng/ul) was injected into the gonads of adult wild-type N2 animals.

  2. To construct the C. elegans plasmid for expression of nsy-1 in AWC neuron, 1348 bp promoter of odr-1 and genomic DNA of nsy-1 was inserted into the pPD49.26-gfp vector. DNA plasmid mixture containing Podr-1::nsy-1::GFP (20 ng/ul) and Podr-1::RFP (50 ng/ul) was injected into the gonads of adult nsy-1(ag3).

  3. To construct the C. elegans plasmid for expression of str-130, 2000 bp promoter of str-130 and 1324 bp genomic DNA of str-130 was inserted into the pPD49.26-mcherry vector. DNA plasmid mixture containing Pstr-130::str-130::mcherry (20 ng/µl) and Podr-1::rfp (50 ng/µl) was injected into the gonads of adult CX3695[str-2::gfp+lin-15(+)].

  4. To construct the C. elegans plasmid for expression of daf-2 in intestine, 2549 bp promoter of ges-1 and 5400 bp cDNA of daf-2 was inserted into the pPD95_77-gfp vector. DNA plasmid mixture containing Pges-1::daf-2::gfp(20 ng/µl) and Podr-1::rfp (50 ng/µl) was injected into the gonads of adult nsy-1(ag3);daf-2(e1370).

  5. To construct the C. elegans plasmid for expression of sid-1 in AWC neuron, 2000 bp promoter of ceh-36 and 2328 bp cDNA of sid-1 was inserted into the pPD49.26-mcherry vector. DNA plasmid mixture containing Pceh-36::sid-1::mcherry (20 ng/µl) and rol-6 (50 ng/µl) was injected into the gonads of adult Pbcf-1::bcf-1::gfp::3xflag;sid-1(qt9).

  6. To construct the C. elegans plasmid for expression of ins-23, 2017 bp promoter of ins-23 and 286 bp genomic DNA of ins-23 was inserted into the pPD49.26-gfp vector. DNA plasmid mixture containing Piins-23::ins-23::gfp (20 ng/µl) and Podr-1::rfp (50 ng/µl) was injected into the gonads of adult wild-type N2 animals.

Generation nsy-1 AWC neuron-specific knockout strain and genotyping

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To construct the C. elegans plasmid for knockout of nsy-1 in AWC neuron, 600 bp promoter of eft-3 was replaced by 1348 bp promoter of odr-1 and nsy-1 sgRNA was also inserted into the same CRISPR-Cas9-sgRNA vector pDD162 (Dickinson et al., 2013). The Cas9 target sites were designed via CRISPR design tool (http://crispor.tefor.net/) and the sgRNA sequence was 5’-GAATTTACGCGTTCGAGAAATGG-3’. Knockout strains were generated by injecting 25 ng/μl Cas9-sgRNA plasmid, 2 μM repair template, co-injection markers include 20 ng/μl dpy-10 Cas9-sgRNA plasmid and 2 μM dpy-10 repair template.

Worms were picked into 10 μl of worm lysis buffer (50 mM KCl, 10 mM Tris–HCl pH 8.0, 2.5 mM MgCl2, 0.45% NP40, 0.45% Tween-20, 0.01% gelactin, 0.2 mg/ml proteinase K), quickly freeze–thaw three times using liquid nitrogen, incubated it at 60°C for 90 min and 95°C for 20 min. 1 µl supernatant was taken and performed for PCR analysis with the following primers: nsy-1: forward 5′-CAAGAGGCAAGTGCAGCATA-3′, reverse 5′-TGACTGTCCCATGCTCTCAC-3′, then digested with NheI endonuclease overnight and identified by DNA agarose electrophoresis.

Preparation of SS

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SS preparation was followed by our published protocol (Geng et al., 2022; Liu and Qi, 2023). Briefly, a standard overnight culture of SS (37℃ in LB broth) was diluted into fresh LB broth (1:100 ratio). SS was then spread onto each NGM plate when the diluted bacteria grew to OD600=0.5.

Analysis of worm’s growth in SS bacteria

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The standard overnight cultured SS was then spread onto 60 mm NGM plate. Worms were grown on E. coli - OP50, and eggs were collected by bleaching and then washing in M9 buffer. Synchronized L1 larvae were obtained by allowing the eggs to hatch in M9 buffer for 12 h. Synchronized L1s were seeded to plates prepared for the specific assay and incubated at 20℃ for 4 days. The developmental conditions were determined by body length.

Food behavior assay

Food avoidance assay

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Food avoidance assay was performed following our published methods (Qi et al., 2017). Briefly, 5 ul of overnight cultured bacteria was seeded on the center of 60 mm NGM plates. About 30–50 synchronized L1 animals were seeded onto the bacterial lawns and cultured at 20℃ for 8 h. The aversion index was determined by N(out of lawn)/N(total).

Food choice assay

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Food choice assay was performed following our published methods (Qi et al., 2017). Briefly, 5 ul of overnight cultured bacteria was seeded on the different side of 60 mm NGM plates. About 300 synchronized L1 animals were seeded onto the center of NGM plates and cultured at 20℃ for 8 h. The food trend index was determined by N (selecting food1)/[N(selecting food1)+N(selecting food2)] and N(selecting food2)/[N(selecting food1)+N(selecting food2)].

Lifespan analysis

Larval lifespan

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Studies of larval lifespan were performed on NGM plates at 20℃ as previously described (Cui et al., 2013). Briefly, L1 staged worms were placed on NGM plates or SS-seeded NGM plates. Worms were scored every day. Prism8 software was used for statistical analysis.

Adult lifespan

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Studies of lifespan were performed on NGM plates at 20℃ as previously described (Kimura et al., 1997).

Briefly, lifespan was begun on day 0 by placing healthy L4 stage hermaphrodites onto OP50 seeded NGM plates. Animals were transferred to a fresh OP50 or SS seeded plates during the reproductive period (approximately the first 10 days) to eliminate self-progeny and every 2 days thereafter. Worms were scored every day. Prism8 software was used for statistical analysis.

Ethyl methanesulfonate (EMS)-induced mutagenesis

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Synchronized L1 animals were grown to the L4 stage on OP50 and then subjected to a 4 h treatment with 0.5% ethyl methanesulfonate (EMS). After treatment, the P0 generation animals were thoroughly washed with M9 solution and transferred to OP50 plates to grow and produce the F1 generation. Groups of 3–4 F1 animals were picked and placed onto new OP50 plates (3–4 F1 worms per plate) to generate the F2 generation through self-fertilization. Once the F2 generation reached adulthood, all the F2 animals were bleached to obtain the next generation of eggs. Synchronized L1 larvae were obtained by allowing the eggs to hatch in M9 buffer for 12 h. The synchronized L1s were then seeded onto SS plates and incubated at 20℃ for 4 days. Candidate mutants that could grow on SS plates were identified. To confirm the suspected mutants, each candidate mutant was picked singly onto OP50 for passaging and growth to adulthood. These adult animals were then bleached to obtain synchronized L1 mutants. The synchronized L1 mutants were seeded onto SS plates to confirm their ability to grow on SS.

Identification of EMS mutants

DNA isolation, library construction, and whole-genome sequencing (WGS) with gene identification were carried out according to the published protocol (Joseph et al., 2018).

Preparation of samples for WGS

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For each backcross, wild-type males were crossed to mutant hermaphrodites, F1 cross-progeny animals were individually cloned and F2 variants and wild-types were isolated under SS feeding condition. The variant strains and wild-type strains were mixed respectively to constitute the ‘DNA-pool’ used as samples for WGS.

WGS of pooled F2 recombinants, homozygous for the mutant phenotype following two outcrosses to wild-type N2 animals, was performed to identify the mutations.

WGS data processing

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For WGS, paired-end libraries were sequenced on an Illumina HiSeq 2000. Fastqc was used to control the quality of raw data, and Trimmomatic was used to filter the data. Bwa was used to construct the index of C. elegans genome and align clean reads to the reference gene sequence (Species: Caenorhabditis_elegans; source: UCSC; reference genome version: WBcel235/ce11). Samtools was used for file format conversion and sorting. Picard was used to remove the duplicate reads and then GATK was used to identify the intervals and realign. The realigned sequence was piled up by Samtools and then inputted to Varscan to call the variants including SNPs and INDELs. Vcflib was used to perform subtraction between the wild-types and variants. The file was annotated by Snpeff, and the candidate genes were finally obtained.

Preparation of samples for RNA sequencing

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RNA-seq was done with three biological replicates that were independently generated, collected, and processed. Adult wild-type (N2) or nsy-1 mutant worms were bleached and then the eggs were incubated in M9 for 18 h to obtain synchronized L1 worms. Synchronized L1 worms were cultured in the NGM plate seeded with OP50 or SS for 4 h at 20°C. L1 worms were then collected for sequencing.

Fluorescence microscopy of C. elegans

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Slides for imaging were prepared by making a fresh flattened 5% agarose pad. Worms were mounted on 5% agar pads in M9 buffer with 5 mM levamisole then sealed beneath a 22 × 22 mm coverglass. Imaging was done using an Olympus BX53 microscope with a DP80 camera. str-2::gfp or bcf-1::gfp expression was measured through imaging.

Microscopy

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The fluorescence photographs were taken using an Olympus BX53 microscope with a DP80 camera. Development statistics were taken using Olympus MVX10 dissecting microscope with a DP80 camera.

Quantification and statistical analysis

Quantification

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Animals were randomly selected for fluorescent photography. The size of transgene worms was photographed using the Nomarski microscope and measured using ImageJ software. ImageJ software was used for quantifying fluorescence intensity of indicated animals, which was then normalized with the control group.

Statistical analysis

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All experiments were performed independently at least three times with similar results. All statistical analyses were performed using the unpaired two-tailed Student’s test. Statistical parameters are presented as mean ± SD, statistical significance (*p<0.05, **p<0.01, ***p<0.001), and ‘n’ (the number of worms counted).

The log-rank (Mantel–Cox) test was used for lifespan assay, and the exact p values of statistics for all survival assays are listed in the figures.

Data availability

Sequencing data have been deposited in CNCB under accession codes PRJCA042026. All data generated or analyzed during this study are included in the manuscript and supporting files; source data files have been provided for all Figures. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request (qb@ynu.edu.cn).

The following data sets were generated

References

Article and author information

Author details

  1. Yating Liu

    Southwest United Graduate School, Yunnan Key Laboratory of Cell Metabolism and Diseases, State Key Laboratory of Conservation and Utilization of Bio-resources in Yunnan, Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, China
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Project administration, Writing – review and editing
    Contributed equally with
    Guojing Tian
    Competing interests
    No competing interests declared

  2. Guojing Tian

    Southwest United Graduate School, Yunnan Key Laboratory of Cell Metabolism and Diseases, State Key Laboratory of Conservation and Utilization of Bio-resources in Yunnan, Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, China
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration
    Contributed equally with
    Yating Liu
    Competing interests
    No competing interests declared

  3. Ziyi Wang

    Southwest United Graduate School, Yunnan Key Laboratory of Cell Metabolism and Diseases, State Key Laboratory of Conservation and Utilization of Bio-resources in Yunnan, Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, China
    Contribution
    Data curation, Investigation, Methodology
    Competing interests
    No competing interests declared

  4. Junkang Zheng

    Southwest United Graduate School, Yunnan Key Laboratory of Cell Metabolism and Diseases, State Key Laboratory of Conservation and Utilization of Bio-resources in Yunnan, Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, China
    Contribution
    Data curation, Investigation, Methodology
    Competing interests
    No competing interests declared

  5. Huimin Liu

    Southwest United Graduate School, Yunnan Key Laboratory of Cell Metabolism and Diseases, State Key Laboratory of Conservation and Utilization of Bio-resources in Yunnan, Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, China
    Contribution
    Data curation, Investigation, Methodology
    Competing interests
    No competing interests declared

  6. Sucheng Zhu

    Southwest United Graduate School, Yunnan Key Laboratory of Cell Metabolism and Diseases, State Key Laboratory of Conservation and Utilization of Bio-resources in Yunnan, Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, China
    Contribution
    Supervision, Methodology
    Competing interests
    No competing interests declared

  7. Zhao Shan

    Southwest United Graduate School, Yunnan Key Laboratory of Cell Metabolism and Diseases, State Key Laboratory of Conservation and Utilization of Bio-resources in Yunnan, Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, China
    Contribution
    Conceptualization, Supervision, Funding acquisition, Writing – original draft, Project administration, Writing – review and editing
    For correspondence
    shanzhaolab@163.com
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5064-1023

  8. Bin Qi

    Southwest United Graduate School, Yunnan Key Laboratory of Cell Metabolism and Diseases, State Key Laboratory of Conservation and Utilization of Bio-resources in Yunnan, Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, China
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Validation, Visualization, Writing – original draft, Project administration, Writing – review and editing
    For correspondence
    qb@ynu.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2261-1550

Funding

Yunnan Province Science and Technology Department (202302AP370005)

  • Bin Qi

Ministry of Science and Technology of the People's Republic of China (2019YFA0803100)

  • Bin Qi

National Natural Science Foundation of China (32071129)

  • Zhao Shan

National Natural Science Foundation of China (32170794)

  • Bin Qi

Yunnan Revitalization Talent Support Program (C619300A086)

  • Zhao Shan

Yunnan Revitalization Talent Support Program (K264202230211)

  • Bin Qi

Yunnan Province Science and Technology Department (202201AT070196)

  • Bin Qi

Ministry of Science and Technology of the People's Republic of China (2019YFA0802100)

  • Bin Qi

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank the Caenorhabditis Genetics Center (CGC) (funded by NIH P40OD010440) for strains. We thank Dr. Huanhu Zhu and Dr. Xiajing Tong (ShanghaiTech University), Dr. Mintie Pu (Yunnan University), Dr. Hongyun Tang (Westlake University), and Dr. Zhiyong Shao (Fudan University) for sharing strains. This work was supported by the Yunnan Provincial Science and Technology Project at Southwest United Graduate School (202302AP370005 to BQ), Yunnan Provincial Science and Technology Project (202201AT070196 to BQ), Yunnan Revitalization Talent Support Program (C619300A086 to ZS, K264202230211 to BQ), Ministry of Science and Technology of the People’s Republic of China (2019YFA0803100, 2019YFA0802100 to BQ), and the National Natural Science Foundation of China (32071129 to ZS, 32170794 to BQ).

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© 2025, Liu, Tian et al.

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  1. Yating Liu
  2. Guojing Tian
  3. Ziyi Wang
  4. Junkang Zheng
  5. Huimin Liu
  6. Sucheng Zhu
  7. Zhao Shan
  8. Bin Qi
(2025)
Neuronal detection triggers systemic digestive shutdown in response to adverse food sources in Caenorhabditis elegans
eLife 14:RP104028.
https://doi.org/10.7554/eLife.104028.3

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