Intestinal pathogens override hunger-driven decision-making via immune regulation of central serotonin signaling in C. elegans

Domain: chemistry
Published: 2026-02-25
Editorial outcome: minor_revision

Abstract
Animals integrate internal states to guide survival-critical decisions, but whether and how intestinal bacteria influence this process by interacting with host metabolic cues remains unclear. Here we show that the intestinal pathogen P. aeruginosa overrides host decision-making in fasted C. elegans by modulating central serotonin (5-HT) signaling. Fasting promotes risk-taking by activating an intestinal energy-sensing pathway that induces the chemoreceptor SRI-36 in ADF 5-HT neurons, sensitizing ADF to food odors and triggering moderate 5-HT release that drives food attraction despite risk. In contrast, intestinal P. aeruginosa reverses this strategy by activating a distinct immune-brain axis that further amplifies SRI-36 expression and ADF sensitivity, leading to excessive 5-HT release that suppresses food attraction and prioritizes safety. These findings reveal a gut-to-brain mechanism by which metabolic and immune signals converge on central 5-HT to reshape behavioral strategies.

Introduction
To survive in dynamic environments filled with both threats and rewards, animals must detect and integrate external sensory cues to make adaptive behavioral choices1,2. This process, known as multisensory threat-reward decision-making, is strongly shaped by internal states, which optimize behavioral responses to meet survival needs. For example, hunger often biases decisions toward risk-taking, prioritizing food acquisition despite potential threats3–6. One effective way internal states influence decision-making is through sensory modulation. Feeding states can alter sensory neuron sensitivity, rapidly shifting sensory perception and behavioral preferences7–9. While such adaptive mechanisms promote survival, maladaptive changes in sensory processing and impaired threat-reward decision-making are hallmarks of neuropsychiatric disorders such as autism, schizophrenia, anxiety, and depression2,10,11. Yet, how internal states regulate sensory processing to guide decision-making remains poorly understood.

Among internal cues, intestinal bacteria are increasingly recognized as potent modulators of brain function and behavior12,13. Intestinal pathogens, in particular, can elicit profound neural changes, including shifts in sensory preferences and the development of anhedonia14,15, a core symptom of neuropsychiatric disorders characterized by the inability to experience reward16. Dysbiosis of the gut microbiota is associated with multiple neuropsychiatric conditions involving impaired decision-making13,17,18. Despite these associations, the mechanisms by which intestinal bacteria influence host decision-making remain elusive. In particular, whether they interact with metabolic states to regulate sensory processing and behavioral choices is unknown.

The nematode C. elegans provides a powerful genetic model for dissecting host-microbe interactions and their impact on neural function and behavior19,20. Its simple intestinal anatomy and compact, well-defined nervous system facilitate mechanistic studies using monoxenic bacterial cultures19,21. C. elegans also exhibits multisensory decision-making behaviors22. For example, when facing a hyperosmotic glycerol barrier placed before the attractive food odor diacetyl, worms must decide whether to risk crossing the potentially lethal barrier to reach the reward6,23. This simple form of decision-making is state-dependent, as hunger enhances risk-taking6,24. Glycerol and diacetyl are detected by the primary sensory neurons ASH and AWA, respectively6,25, and downstream interneurons integrate these inputs to drive behavior26. However, whether intestinal pathogens alter these processes and how they may interact with metabolic states to regulate behavioral choices remains unknown.

Here, we show that the intestinal pathogen P. aeruginosa overrides hunger-driven decision-making in C. elegans by modulating central serotonin (5-HT) signaling through an immune-brain axis. Mechanistically, fasting promotes risk-taking by activating the intestinal energy sensor AAK-2/AMPK, which promotes DAF-16/FOXO-dependent release of the insulin-like peptide (ILP) INS-37 from intestinal epithelial cells (IECs). INS-37 then engages the canonical DAF-2-DAF-16 insulin/IGF-1 signaling (IIS) cascade in ADF 5-HT neurons. This FOXO-to-FOXO signaling induces expression of the chemoreceptor SRI-36, conferring diacetyl sensitivity to ADF, which are otherwise insensitive in the well-fed state. Diacetyl-evoked ADF activation triggers moderate 5-HT release that inhibits AIB and activates AUA interneurons via the 5-HT receptors SER-4 and LGC-50, respectively, promoting diacetyl attraction despite the associated risk. In contrast, intestinal P. aeruginosa subverts this hunger-driven decision-making by activating the intestinal PMK-2/p38 MAPK immune pathway, which induces a distinct ILP, INS-7, to further amplify SRI-36 expression and ADF responsiveness through the same FOXO-to-FOXO axis. This leads to excessive 5-HT release that predominantly inhibits AIA and AIY interneurons via the 5-HT receptor MOD-1, suppressing diacetyl attraction and prioritizing safety.

Together, these findings establish P. aeruginosa as an intestinal pathogen capable of overriding host decision-making through immune modulation of central 5-HT signaling, and identify central 5-HT as a convergence target for intestinal metabolic and immune cues. Our study provides a mechanistic framework for how intestinal microbes and internal physiological states dynamically modulate brain function and behavioral decisions.

Results
Intestinal pathogen P. aeruginosa reverses hunger-driven decision-making via ADF 5-HT neurons in C. elegans
To investigate how intestinal pathogens influence host decision-making, we employed a multisensory behavioral choice assay in C. elegans6,23. In this assay, worms are attracted to the food odor diacetyl but must decide whether to cross a hyperosmotic glycerol barrier to reach it (Supplementary Fig. 1a). We tested three paradigmatic human pathogenic bacteria, Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), and enteropathogenic E. coli (EPEC). However, none of these pathogens altered the behavioral choice under well-fed conditions, as escape percentages remained comparable to those of control worms fed nonpathogenic E. coli (Supplementary Fig. 1b).

Because hunger is known to promote risk-taking3–6, we adapted the assay by fasting worms before the choice test (Fig. 1a). Fasting increased escape probability in a time-dependent manner across all glycerol concentrations tested, with the strongest effect observed after 6 h of fasting at 3 M glycerol (Supplementary Fig. 1c), which was selected for subsequent experiments. Under these conditions, P. aeruginosa infection significantly reduced escape probability compared to E. coli, returning it to levels similar to well-fed controls, whereas S. aureus and EPEC had no effect (Fig. 1b). In contrast, P. aeruginosa did not affect unisensory decision-making, as chemotaxis toward diacetyl (in the absence of glycerol) and escape from glycerol (in the absence of diacetyl) remained unchanged (Supplementary Fig. 1d, e). Locomotor activity was also unaffected (Supplementary Fig. 1f). These results suggest that P. aeruginosa selectively reverses hunger-driven risky decision-making in C. elegans. Similar escape suppression occurred after 4 or 24 h of infection (Supplementary Fig. 1g), with 4 h chosen for subsequent experiments.

Previous studies have suggested that P. aeruginosa metabolites may modulate C. elegans behavior via chemosensory neurons27,28. However, exposure to P. aeruginosa supernatant, odors, heat-killed cells, or a nonpathogenic mutant gacA(−) did not affect fasting-induced escape (Fig. 1c). In contrast, infection with live cells alone was sufficient (Fig. 1c), and enhancing virulence by supplementing the medium with 0.35% peptone29 increased P. aeruginosa colonization and escape suppression (Fig. 1d and Supplementary Fig. 1h). Knockdown of nol-6, which reduces intestinal colonization of P. aeruginosa30 (Fig. 1e), attenuated its effect on escape (Fig. 1f). Consistent with the similar escape behavior observed at 4 h and 24 h of infection, intestinal P. aeruginosa colonization did not differ significantly between these time points (Supplementary Fig. 1g, i). These results suggest that intestinal colonization of virulent P. aeruginosa is required to suppress hunger-driven decision-making.

5-HT and dopamine are key neuromodulators that regulate cognitive processes, including threat and reward processing22,31. Loss of the dopamine biosynthetic gene cat-232 had no effect on escape in the fasted or infected state (Fig. 1g). However, loss of the 5-HT biosynthetic gene tph-132 reduced fasting-induced escape and reversed the P. aeruginosa-induced suppression of escape (Fig. 1h). Additionally, mod-5(n822) mutants, which have elevated extracellular 5-HT due to impaired reuptake33, showed exaggerated behavioral responses to both hunger and infection (Fig. 1i). These results suggest that both hunger and P. aeruginosa regulate decision-making through 5-HT signaling.

To identify the relevant 5-HT neurons, we silenced tph-1-expressing neurons NSM, ADF, or HSN34 using the histamine-gated chloride channel HisCl135. Silencing NSM or HSN had no effect (Fig. 1k, l), but silencing ADF significantly impaired both fasting-induced escape and P. aeruginosa-mediated suppression (Fig. 1j). Consistently, expressing wild-type tph-1 in ADF, but not NSM, fully rescued the effects of both fasting and P. aeruginosa (Fig. 1h). Together, these results suggest that hunger promotes risky decision-making, while P. aeruginosa overrides this decision, both via ADF 5-HT neurons.

In addition to 5-HT, ADF neurons are also cholinergic36. However, ADF-specific unc-17 RNAi, which reduces vesicular acetylcholine release37, did not significantly affect escape in either the fasted or infected state (Supplementary Fig. 1j). These results suggest that acetylcholine release from ADF is not required for the state-dependent regulation of decision-making, although it may contribute to other ADF-mediated functions.

ADF neurons act as primary olfactory sensory neurons for diacetyl in fasted and infected states
We next investigate how hunger and P. aeruginosa infection influence decision-making via 5-HT signaling. ASH and AWA sensory neurons, which detect glycerol and diacetyl respectively6,25, form direct synaptic connections with ADF38 (Supplementary Fig. 2a). We hypothesized that changes in these upstream neurons might influence ADF responses under different internal states. Indeed, fasting reduced glycerol-evoked calcium responses in ASH and enhanced diacetyl-evoked responses in AWA (Supplementary Fig. 2b, c). However, P. aeruginosa infection did not further alter ASH or AWA responses in fasted worms (Supplementary Fig. 2b, c). Silencing AWA with HisCl reduced escape in both well-fed and fasted states, consistent with its role in sensing diacetyl, but fasted worms still exhibited substantial escape probability compared to well-fed controls (Supplementary Fig. 2d). These results suggest that while ASH and AWA sensory responses are modulated by hunger, they are not the primary sites through which P. aeruginosa infection overrides hunger-driven decision-making.

ADF neurons are 5-HT sensory neurons that respond to noxious stimuli in well-fed worms39 and to food odors under dietary restriction40. In a transgenic strain expressing GCaMP6 in ADF, which exhibited normal escape behavior compared to wild type (Supplementary Fig. 2e), ADF showed no response to glycerol but were robustly activated by diacetyl (Fig. 2a, b), with responses enhanced by fasting and further amplified by P. aeruginosa infection (Fig. 2b). In unc-13(e51) mutants, which are defective in neurotransmitter release41, diacetyl-evoked ADF responses were absent in well-fed worms but persisted in fasted and infected states, albeit at a reduced amplitude (Fig. 2c). In contrast, unc-31(e169) mutants, which are defective in neuropeptide release42,43, showed no such defect (Fig. 2c), Moreover, in odr-7(ky4) mutants, where AWA neurons were eliminated44, and in odr-7(ky4);unc-13(e51) double mutants, ADF responses to diacetyl were abolished in the well-fed state but retained in both fasted and infected states (Fig. 2d). These results suggest that while ADF neurons receive synaptic input from AWA in response to diacetyl stimulation, they function as primary olfactory neurons for diacetyl during fasting and P. aeruginosa infection, independent of synaptic input.

Olfactory detection in C. elegans is mediated by G-protein-coupled receptors (GPCRs) and downstream transduction channels45. ODR-3/Gα mediates food odor sensation in ADF neurons40, and the TRPV channel subunits OCR-2 and OSM-9, which are coexpressed in ADF, likely function downstream of GPCR signaling45. In odr-7(ky4) mutants, mutations in odr-3, ocr-2, or osm-9 abolished ADF responses to diacetyl in both fasted and infected worms, and ADF-specific expression of the corresponding wild-type genes rescued the defect (Fig. 2e). These results suggest that an unidentified GPCR, acting upstream of the ODR-3-OCR-2/OSM-9 signaling pathway, mediates ADF responses to diacetyl during fasting and P. aeruginosa infection.

Hunger- and infection-induced SRI-36 acts as a diacetyl receptor in ADF neurons
To identify the GPCR responsible for ADF diacetyl sensitivity, we first tested ODR-10, the only known diacetyl receptor, which is exclusively expressed in AWA neurons of well-fed worms46. However, ADF responses to diacetyl remained comparable between unc-13(e51) and unc-13(e51);odr-10(ky32) mutants across all states (Fig. 3a), indicating that ODR-10 is not required for ADF sensitivity in the absence of synaptic input.

We next analyzed published RNA-sequencing datasets for chemosensory GPCRs upregulated during fasting or P. aeruginosa infection. We identified 126 candidate GPCRs induced by fasting (Fasted vs. Well-fed)47 and 1,667 genes upregulated by P. aeruginosa exposure (P. aeruginosa vs. E. coli)48, with four GPCRs, sri-36, srr-6, str-163, and srap-1, shared between comparisons (Fig. 3b). Screening these genes in P. aeruginosa-infected odr-7(ky4) mutants revealed that ADF-specific RNAi of sri-36, but not the other candidates, abolished ADF calcium responses to diacetyl (Fig. 3c). This effect was confirmed in fasted odr-7(ky4) mutants (Fig. 3d).

To further validate the role of SRI-36, we generated a CRISPR deletional allele, sri-36(plc921) (Supplementary Fig. 3a), which eliminated ADF responses to diacetyl in both fasted and infected odr-7(ky4) mutants (Fig. 3e). This defect was rescued by ADF-specific expression of wild-type sri-36 (Fig. 3e). The deletion also impaired escape in both fasted and infected worms, which was rescued by ADF-specific expression of wild-type sri-36 and recapitulated by ADF-specific RNAi (Fig. 3f). A Psri-36::GFP transcriptional reporter was undetectable in well-fed worms but strongly induced in ADF during fasting and further enhanced by P. aeruginosa infection (Fig. 3g and Supplementary Fig. 3b). Consistently, a CRISPR knock-in reporter, SRI-36::mNeonGreen, with mNeonGreen inserted at the C-terminus of the endogenous locus, showed a similar ADF-specific induction pattern (Supplementary Fig. 3c). To test whether receptor abundance modulates ADF sensitivity, we expressed SRI-36 at two different levels in sri-36(plc921) mutants by injecting 5 ng/µl (low) or 75 ng/µl (high) of Psrh-142::sri-36::mStrawberry plasmid (Supplementary Fig. 3d). In the fasted state, low-level expression restored ADF diacetyl responses and escape to control levels (Fig. 3e, f). In contrast, high-level expression enhanced ADF diacetyl responses and suppressed escape, phenocopying the behavioral pattern observed during P. aeruginosa infection (Fig. 3e, f). These results suggest that SRI-36 is induced in ADF by hunger and infection and is required for diacetyl sensation, and that its expression level tunes ADF sensitivity and behavioral output.

To test whether SRI-36 functions as a diacetyl receptor, we performed multiple receptor validation assays29,49. First, both an SRI-36::GFP translational fusion and the CRISPR-generated endogenous SRI-36::mNeonGreen fusion protein localized to the sensory cilia of ADF (Fig. 3h and Supplementary Fig. 3c), supporting its role in detecting environmental odors. Second, ectopic expression of sri-36 in ASH nociceptive neurons, which normally do not respond to the attractive food odor diacetyl, conferred robust diacetyl-evoked calcium responses in ASH (Supplementary Fig. 3e), demonstrating that SRI-36 is sufficient to mediate diacetyl sensitivity. Third, because ASH activation drives avoidance25, we assessed behavioral consequences using a two-choice chemotaxis quadrant assay29. Ectopic sri-36 expression in ASH significantly reduced diacetyl attraction (Supplementary Fig. 3f). Notably, in odr-7(ky4) mutants, ASH-expressed sri-36 even converted diacetyl attraction to aversion (Supplementary Fig. 3f), further supporting the role of SRI-36 in mediating diacetyl sensation. Together, these results establish SRI-36 as a diacetyl receptor or essential receptor subunit in ADF neurons.

Hunger- and infection-induced SRI-36 expression in ADF requires intestinal AAK-2/AMPK and PMK-2/p38 MAPK pathways
To investigate how fasting and P. aeruginosa infection regulate ADF diacetyl sensitivity, we examined the roles of the AAK-2/AMPK energy sensor and the PMK-1/p38 MAPK immune pathway. AMPK is a key metabolic sensor that responds to nutrient deprivation40,50. In C. elegans, AAK-2/AMPK phosphorylates and activates the transcription factor DAF-16/FOXO, driving DAF-16-dependent gene expression40,51. In fasted odr-7(ky4) mutants, aak-2 deletion abolished diacetyl-evoked calcium responses in ADF, which were restored by intestinal expression of wild-type aak-2 and recapitulated by intestine-specific RNAi of aak-2 (Fig. 4a). Similarly, intestine-specific RNAi of daf-16 in odr-7(ky4) mutants reduced ADF responses to levels comparable to odr-7(ky4);aak-2(ok524) mutants (Fig. 4a). Fasting-induced nuclear translocation of DAF-16 was also reduced by intestine-specific RNAi of aak-2 (Fig. 4b), whereas intestinal overexpression of a constitutively nuclear-localized DAF-16 (DAF-16aAM)52 restored ADF responses in fasted odr-7(ky4);aak-2(ok524) mutants (Fig. 4a). The intestine-specific RNAi of aak-2 and daf-16 produced similar results in the systemic RNAi-defective sid-1(qt9) mutant background53 (Fig. 4a, b), confirming tissue specificity. These results suggest that AAK-2 activates DAF-16 in IECs to drive ADF diacetyl sensitivity during fasting.

We next examined the role of the PMK-1/p38 MAPK immune pathway, which cooperates with DAF-16 in epithelial immunity against P. aeruginosa48,54. Given that p38 MAPK can activate AKT and antagonize DAF-16 nuclear translocation55, we hypothesized that P. aeruginosa modulates ADF diacetyl sensitivity via p38-mediated inhibition of DAF-16. Indeed, in infected odr-7(ky4) mutants, ADF responses were abolished by mutations or intestine-specific RNAi of p38 MAPK pathway genes nsy-1 or sek-1 (Fig. 4c). Unexpectedly, deletion of pmk-1 had no effect (Fig. 4c). The C. elegans genome encodes three p38 homologs, PMK-1, PMK-2, and PMK-3. Intestine-specific RNAi of pmk-2 eliminated ADF responses in odr-7(ky4) mutants, whereas deletion of pmk-3 had no effect (Fig. 4c). As previously reported54, P. aeruginosa infection significantly inhibited DAF-16 nuclear translocation (Fig. 4b). This inhibition was reversed by intestine-specific RNAi of pmk-2 (Fig. 4b). Notably, intestinal DAF-16aAM overexpression suppressed ADF responses in odr-7(ky4) mutants, with no additive effect when combined with intestine-specific RNAi of pmk-2 (Fig. 4c). Comparable intestine-specific RNAi effects were observed in the sid-1(qt9) background (Fig. 4b, c), confirming tissue specificity. These results suggest that PMK-2 inhibits DAF-16 in IECs to suppress ADF diacetyl sensitivity during P. aeruginosa infection.

We then asked whether these pathways regulate sri-36 expression in ADF and influence decision-making. Intestine-specific RNAi of aak-2 or daf-16 similarly reduced Psri-36::GFP fluorescence and escape in fasted worms (Fig. 4d, e). Simultaneous RNAi of both genes produced no additive effect on escape (Fig. 4e). In infected worms, intestine-specific RNAi of pmk-2 or overexpression of DAF-16aAM similarly reduced Psri-36::GFP expression and impaired escape (Fig. 4f, g). RNAi of pmk-2 in the presence of DAF-16aAM also produced no additive effect on escape (Fig. 4g). Comparable intestine-specific RNAi effects were observed in the sid-1(qt9) background (Fig. 4d–g), confirming tissue specificity. These results suggest that both the AAK-2/AMPK energy-sensing pathway and the PMK-2/p38 MAPK immune pathway remotely regulate ADF sri-36 expression via DAF-16/FOXO from the intestine.

Intestine-released ILPs regulate SRI-36 expression via the IIS pathway in ADF
To investigate how intestinal signals regulate SRI-36 expression in ADF, we considered endocrine signaling, a conserved mechanism for gut-to-brain communication24,56. The C. elegans genome encodes three major neuropeptide families, FLPs, NLPs, and ILPs57. Intestine-specific RNAi of hid-1, a gene required for intestinal neuropeptide sorting and secretion58, or deletion of egl-3, encoding a proprotein convertase essential for neuropeptide maturation57, abolished ADF responses in both fasted and infected odr-7(ky4) mutants (Fig. 5a). Comparable intestine-specific RNAi effects were observed in the sid-1(qt9) background (Fig. 5a), confirming tissue specificity. Similarly, ADF-specific RNAi of daf-2, encoding the sole ILP receptor in C. elegans59, eliminated ADF responses (Fig. 5b). These results suggest that intestinal ILPs act via DAF-2 in ADF to promote diacetyl sensitivity.

The C. elegans genome encodes 40 ILPs57,59. To identify the specific ILPs involved, we performed a feeding RNAi screen using the intestine-specific RNAi strain VP303 and found that INS-37 and INS-7 selectively regulated ADF responses in fasted and infected worms, respectively (Supplementary Fig. 4). These effects were confirmed by intestine-specific RNAi of ins-37 or ins-7 via plasmid injection (Supplementary Fig. 4c, f). Additionally, intestine-specific RNAi of ins-37 and deletion of ins-7 abolished ADF responses in fasted and infected odr-7(ky4) mutants, respectively (Fig. 5c, d). The defect of ins-7(tm2001) was rescued by intestine-specific expression of wild-type ins-7 and recapitulated by intestine-specific RNAi (Fig. 5d). Furthermore, fasting-induced expression of the transcriptional reporter Pins-37::GFP, while P. aeruginosa infection suppressed it (Fig. 5e). Intestine-specific RNAi of aak-2, daf-16, or both genes reduced Pins-37::GFP expression similarly in fasted worms (Fig. 5f). In contrast, P. aeruginosa infection significantly increased Pins-7::GFP expression, which was blocked by intestine-specific RNAi of pmk-2 or overexpression of DAF-16aAM, with no additive effect when combined (Fig. 5g). Comparable intestine-specific RNAi effects were observed in the sid-1(qt9) background (Fig. 5c, d, f, g), confirming tissue specificity. Notably, Pins-7::GFP expression did not differ significantly between 4 h and 24 h of infection but was enhanced by 0.35% peptone, consistent with escape behavior (Supplementary Fig. 5a, b). Among other tested bacteria, although S. aureus also colonized the intestine, it did not induce Pins-7::GFP expression (Supplementary Fig. 5e, f). These results are consistent with a model in which hunger and P. aeruginosa infection induce intestinal INS-37 and INS-7 release, respectively, via AAK-2-DAF-16 and PMK-2-DAF-16 pathways, which in turn regulate ADF diacetyl sensitivity.

To test the specificity of these pathways, we performed reciprocal reporter assays. Fasting-induced Pins-37::GFP expression was not altered by intestinal pmk-2 RNAi (Supplementary Fig. 5c). In contrast, P. aeruginosa infection-induced Pins-7::GFP expression was further enhanced in aak-2 RNAi worms after 1 h and 2 h of infection, but not at 4 h, suggesting a transient peak of infection-induced ins-7 expression (Supplementary Fig. 5d). These results indicate that pmk-2 signaling is not required for fasting-induced ins-37 expression, whereas aak-2 normally acts to dampen ins-7 induction during infection. Thus, while aak-2 and pmk-2 serve as primary mediators of fasting- and infection-induced responses, respectively, these pathways also exhibit limited cross-talk.

To determine whether INS-37 and INS-7 regulate SRI-36 expression, we analyzed Psri-36::GFP fluorescence in ADF and escape behavior. In fasted worms, intestine-specific ins-37 RNAi or ADF-specific daf-2 RNAi similarly reduced Psri-36::GFP expression and escape, with no additive effect when combined (Fig. 5h, i). In infected worms, intestine-specific ins-7 RNAi or ADF-specific daf-2 RNAi similarly suppressed Psri-36::GFP expression and enhanced escape, again with no additive effect when combined (Fig. 5h, i). These results demonstrate that intestine-released INS-37 and INS-7 act through DAF-2 in ADF to regulate SRI-36 expression and decision-making. Consistently, P. aeruginosa also induced Pins-7::GFP expression and weak Psri-36::GFP expression in well-fed worms (Supplementary Fig. 5g, h). However, because P. aeruginosa did not affect escape in well-fed worms (Supplementary Fig. 5i), these results suggest that while P. aeruginosa activates intestinal immune signaling in both well-fed and fasted worms; its behavioral suppression is most pronounced under fasting, when hunger-driven risk-taking is elevated.

To dissect transcriptional mechanisms downstream of DAF-2, we examined known effectors of the IIS pathway. DAF-16/FOXO, HSF-1/HSF, SKN-1/Nrf2, PHA-4/FOXA, and DAF-12 are generally inhibited by DAF-2, while PQM-1 is activated by DAF-259,60. Interestingly, the upstream regulatory sequence of sri-36 contains predicted binding sites60 for both DAF-16 and PQM-1 (Supplementary Fig. 6a). In well-fed odr-7(ky4) mutants, ADF-specific RNAi of daf-16, but not other IIS effectors, induced robust diacetyl responses in ADF (Supplementary Fig. 6b). In infected worms, ADF-specific RNAi of pqm-1 had no effect (Supplementary Fig. 6c). These results suggest that DAF-16 represses sri-36 expression in ADF.

Consistently, ADF-specific daf-16 RNAi induced Psri-36::GFP expression in well-fed worms, while ADF-specific DAF-16aAM overexpression suppressed Psri-36::GFP expression and impaired escape in both fasted and infected worms (Fig. 5j, k). These results support a model in which DAF-16 acts cell-autonomously in ADF to inhibit sri-36 expression, and that ILP-DAF-2 signaling relieves this inhibition to drive sensory plasticity during hunger and infection.

AIB/AUA and AIA/AIY interneurons act downstream of ADF to regulate behavioral choices
We next sought to identify the interneurons that act downstream of ADF to mediate state-dependent decision-making. ADF neurons form synapses with multiple interneurons, including AIA, AIB, AIY, AIZ, AUA, and RIA38, with AIA, AIB, AIY, and AIZ serving as first-layer chemosensory interneurons26. In fasted worms, silencing AIB alone, AIB/AIZ, or AUA/URX using HisCl1 significantly reduced escape, whereas silencing AIY/AIZ, URX alone, or other interneurons had no effect (Fig. 6a). Notably, simultaneous silencing of AIB/AUA/URX further decreased escape compared to silencing AIB or AUA/URX alone (Fig. 6a). In infected worms, silencing AIA or AIY significantly increased escape, and the effect was amplified when both were silenced simultaneously (Fig. 6a). To investigate how ADF neurons regulate these interneurons, we performed calcium imaging. In fasted odr-7(ky4) mutants, diacetyl exposure inhibited AIB and activated AUA (Fig. 6b). In infected mutants, diacetyl inhibited both AIA and AIY (Fig. 6c). Silencing ADF using HisCl1 abolished diacetyl-evoked responses in these interneurons (Fig. 6b, c), whereas silencing AIB/AUA or AIA/AIY had no effect on ADF calcium responses (Supplementary Fig. 7a). These results suggest that ADF neurons regulate decision-making through distinct interneuron pathways depending on internal states. In fasted worms, ADF neurons promote risk-taking by inhibiting AIB and activating AUA, whereas in infected worms, they suppress risk-taking by inhibiting AIA and AIY. This is consistent with established roles of AIB in promoting avoidance, AUA in suppressing avoidance, and AIA/AIY in promoting attraction26,61.

We next identified the 5-HT receptors mediating ADF’s effects on these interneurons. Among the six known C. elegans 5-HT receptors62, deletion of ser-4 and lgc-50 significantly reduced escape in fasted worms, while deletion of mod-1 increased escape in infected worms (Supplementary Fig. 7b). ser-4, lgc-50, and mod-1 encode a Gi/o-coupled inhibitory receptor, a 5-HT-gated chloride channel, and a 5-HT-gated cation channel, respectively62. Based on the behavioral roles and activity patterns of AIB, AUA, AIA, and AIY (Fig. 6a–c), we hypothesized that SER-4, LGC-50, and MOD-1 function in AIB, AUA, and AIA/AIY, respectively. Indeed, neuron-specific expression of the corresponding wild-type receptor genes in these interneurons rescued the behavioral phenotypes of the respective mutants (Fig. 6d), consistent with their known expression patterns62. Furthermore, neuron-specific RNAi of ser-4, lgc-50, and mod-1 abolished exogenous 5-HT-evoked calcium responses in AIB, AUA, and AIA/AIY, respectively (Fig. 6e, f). These results identify SER-4, LGC-50, and MOD-1 as key 5-HT receptors that relay ADF 5-HT output to distinct interneurons.

Finally, we tested whether direct modulation of ADF activity is sufficient to shift decision-making. Because diacetyl evoked stronger ADF activation in infected worms than in fasted worms (Fig. 2), we hypothesized that P. aeruginosa infection increases ADF 5-HT release. Indeed, infected odr-7(ky4) mutants exhibited significantly stronger synapto-pHluorin (SNB-1::pHluorin, a synaptic vesicle release reporter63) signals in ADF upon diacetyl stimulation than fasted worms (Fig. 6g), demonstrating enhanced 5-HT release during infection. Consistent with bidirectional control by SRI-36, in fasted sri-36(plc921) mutants, low-level SRI-36 expression (5 ng/µl) restored moderate 5-HT release to a level similar to fasted controls, whereas high-level expression (75 ng/µl) enhanced 5-HT release to a level comparable to P. aeruginosa-infected controls (Fig. 6g). Chemogenetic manipulation of ADF activity further supported this bidirectional control. Enhancing ADF activity in fasted worms using TRPV1, a mammalian cation channel activated by capsaicin64, reduced escape in a dose-dependent manner, an effect abolished by AIA/AIY-specific mod-1 RNAi (Fig. 6h). Conversely, chemogenetically suppressing ADF activity in infected worms using HisCl1 increased escape in a dose-dependent fashion (Fig. 6i). Notable, histamine concentrations above 3 mM reversed this trend and again suppressed escape, consistent with ADF promoting risk-taking at moderate activity levels but suppressing it when excessively activated. This bidirectional effect was attenuated by RNAi of ser-4 in AIB and lgc-50 in AUA (Fig. 6i). Together, these results demonstrate that tuning ADF activity is sufficient to switch behavioral strategies, with moderate ADF activation promoting risk-taking through AIB/AUA and strong activation suppressing it via AIA/AIY.

Methods
Bacterial strains and culture
E. coli OP50, P. aeruginosa PA14, S. aureus NCTC 8325, and EPEC were cultured under conditions optimized for their growth and virulence88,89. E. coli, P. aeruginosa, and EPEC were grown in LB medium, while S. aureus was cultured in tryptic soy broth (Hopebio) supplemented with 10 µg/ml nalidixic acid (Macklin) to optimize growth and prevent overgrowth of contaminants. P. aeruginosa and S. aureus cultures were maintained under gentle shaking. All bacteria were grown overnight at 37 °C before seeding onto agar plates. E. coli was plated on nematode growth medium (NGM), P. aeruginosa on NGM supplemented with 0.25% peptone (Aobox) to support robust virulence factor production while maintaining animal viability for behavioral assays. S. aureus cultures were diluted 1:5 before plating on tryptic soy agar (Hopebio) containing 10 µg/ml nalidixic acid (Macklin). EPEC was plated on modified NGM containing 2 mg/ml tryptophan (Aladdin) to enhance its virulence89. Plates seeded with E. coli and EPEC were incubated at 37 °C for 20 h, P. aeruginosa plates at 37 °C for 24 h followed by 6 h at 25 °C, and S. aureus plates at 37 °C for 6–8 h. All plates were equilibrated at 25 °C for 1–2 h before use. The strains used in this study are listed in Supplementary Data 1.

C. elegans strains and culture
C. elegans strains were raised on NGM plates seeded with E. coli OP50 at 22 °C. Transgenic lines were generated by microinjection of plasmid DNA, and at least two independent lines were tested for confirmation. Integrated transgenic strains were created using gamma irradiation and outcrossed at least three times before use. Unless otherwise noted, all experiments used young adult hermaphrodites. Investigators were not blinded to genotype or condition. The strains used in this study are listed in Supplementary Data 1.

Mutant rescue and RNAi
Mutant rescue was achieved by expressing wild-type cDNAs under cell-specific promoters. RNAi knockdown was performed by co-injecting two plasmids encoding complementary sense and antisense mRNA fragments of the target gene. To confirm tissue specificity and exclude systemic RNAi effects, intestine-specific RNAi experiments (aak-2, daf-16, nsy-1, sek-1, pmk-2, hid-1, ins-37, and ins-7) were repeated in the systemic RNAi-defective sid-1(qt9) mutant background, in which dsRNA spreading is abolished53. Promoters used included Pges-1 (IECs)49, Pceh-2 (NSM)90, Psrh-142 (ADF)90, Pclh-3 (HSN)91, Pgcy-28 (AIA)92, Pttx-3 (AIY)92, Pnpr-9 (AIB)92, Podr-2 (AIB/AIZ)28, Pser-2prom2 (AIY/AIZ)62, Pflp-8 (AUA/URX)93, Pgcy-36 (URX)93, Pglr-3 (RIA)92, Psra-6 (ASH)94, and Pgpa-6 (AWA)95. All cDNAs and RNAi target sequences were cloned from a Bristol N2 cDNA library. Primers (Tsingke Biotechnology Co., Ltd.) are listed in Supplementary Table 1.

CRISPR-Cas9 genome editing
To generate the sri-36(plc921) deletion allele, two sgRNAs (5′ region: AGTACCTGATAAGCCAGACA; 3′ region: TATAATACCAAGAGTGGCCC) were cloned into the pDD162 (Peft-3::Cas9+empty sgRNA) vector. The resulting plasmids (20 ng/µl each) were co-injected into young adult hermaphrodites together with Pmyo-2::mStrawberry (25 ng/µl) as a co-injection marker. F1 progeny were screened by PCR for deletion events, and homozygous mutants were confirmed by sequencing. To generate the endogenous sri-36::mNeonGreen knock-in reporter, three sgRNAs (GAAATTTGTAATCAGAAAGT; TAACTTTTACAATCCCATAT; ACTCCATTCGTCTGTCAATG) targeting the sri-36 C-terminus were used together with a donor repair plasmid containing the mNeonGreen coding sequence flanked by ~1 kb homology arms. The injection mixture contained 5 ng/µl sgRNA-Cas9 plasmids, 25 ng/µl donor plasmid, and 25 ng/µl Pmyo-2::mCherry co-injection marker. Correct knock-in events were identified by PCR and verified by sequencing. All CRISPR-edited strains were outcrossed three times before use.

RNAi screen of intestine-released ILPs
Intestine-specific RNAi of ILP genes was performed by feeding C. elegans strain VP303 with E. coli HT115 carrying L4440 plasmids containing target gene fragments96. Briefly, HT115 was cultured overnight in LB with 100 µg/ml ampicillin (Biosharp) at 37 °C, seeded onto NGM RNAi plates supplemented with 100 µg/ml ampicillin and 5 mM IPTG (Biotopped), and incubated at 37 °C for 12–14 h. Three L4-stage VP303 worms were transferred to each plate at 20 °C until their progeny reached young adulthood. rab-5 RNAi was used as a positive control.

Behavioral analysis
All behavioral assays were conducted at 22 °C. Spontaneous locomotion was recorded and analyzed using the Track-A-Worm automated tracking system (version 2.0)23. A single young adult was transferred to the center of a 6-cm NGM plate, allowed to recover for 30 s, and recorded for 1 min at 15 fps.

For behavioral choice assays23, 6-cm choice plates were prepared by drawing a 1-cm glycerol ring (10 µl, 3 M) and placing two diacetyl spots (1 µl of 1:1000 diacetyl mixed with 1 µl of 0.1 M NaN3) near the plate edges. Synchronized worms were cultured on E. coli OP50 until young adulthood and then transferred to plates seeded with different bacterial strains. After 4 h, worms were washed with M9 buffer, and 10–15 worms were placed at the center of each choice plate. The number of worms at the diacetyl spots was counted after 15 min. For fasting assays, worms were deprived of food on unseeded NGM plates for the indicated time before the choice test. NGM plates containing histamine (Sigma-Aldrich) or capsaicin (Aladdin) were prepared by adding the chemicals at their final concentrations, and then seeded with 200 µl of bacteria pre-mixed with the same concentration of histamine or capsaicin.

Unisensory avoidance assays were performed using glycerol-only plates, and worms outside the glycerol ring were counted after 15 min23. Unisensory attraction assays were performed by dividing plates into four equal quadrants (A–D)23. Diacetyl (1 µl, 1:1000) and control water (1 µl) were applied to opposite edges along with NaN3. Worms were transferred to the center and counted after 15 min. Chemotaxis index = (worms in A − worms in D)/total worms × 100%.

For two-choice chemotaxis quadrant assays29, 9-cm quadrant plates were divided into four sections, with opposite quadrants containing either diacetyl (20 µl, 1:1000) or control water (20 µl). 100–150 M9-washed worms were placed in the center. After 15 min, plates were frozen at −20 °C for 10 min to stop movement, and worms in diacetyl and control quadrants were counted. Chemotaxis index = (worms in diacetyl − worms in control)/total worms × 100%. A positive index indicates attraction to diacetyl.

Imaging
Images of the SRI-36::GFP translational fusion and the CRISPR knock-in SRI-36::mNeonGreen fusion protein were acquired using an Olympus IXplore Spin SR confocal microscope with a 100×/1.45 NA oil objective. All other fluorescence images were captured with a Nikon Ts2R inverted microscope equipped with an Mshot MD60 CCD camera and analyzed using the Mshot Image Analysis System (version 1.1). Acquisition settings were kept constant within each experiment. For DAF-16 subcellular localization analysis, strains co-expressing Pdaf-16::daf-16a/b::GFP and the IEC nuclear marker Pges-1::NLS::mCherry were used. The percentage of GFP-labeled IEC nuclei was counted per animal. For calcium and pHluorin imaging, young adults were immobilized on coverslips using Vetbond Tissue Adhesive (3M Company) in bath solution (140 mM NaCl, 5 mM KCl, 5 mM CaCl2, 5 mM MgCl2, 11 mM dextrose, 5 mM HEPES, pH 7.2, 320 mOsm) and imaged at 1 fps for 2 min. Diacetyl or glycerol was pressure-ejected (2 psi, 30 s) near the nose using an Eppendorf FemtoJet 4i microinjector. For exogenous 5-HT stimulation, because the targeted interneurons (AIA, AIY, AIB, and AUA) are located deep within the head and inaccessible to bath-applied 5-HT, a longitudinal incision was made along the glued region to expose neuron somas. 5-HT was then applied directly to neuron somas using pressure ejections (2 psi, 30 s), ensuring spatial precision, reproducibility, and consistent stimulation of the relevant interneurons.

Quantification of intestinal bacteria accumulation
To quantify the intestinal accumulation of PA14-dsRed97, full-lawn plates were prepared by spreading an overnight culture of PA14-dsRed. Young adult hermaphrodites grown on E. coli OP50 were transferred onto these plates and exposed for 4 h, then deprived of food on unseeded NGM plates for 6 h. Worms were washed with M9 buffer to remove external bacteria and imaged using the Nikon Ts2R inverted microscope.

Intestinal accumulation of E. coli, S. aureus, EPEC, and PA14-gacA(−) was quantified using colony-forming unit (CFU) assays48. Worms were similarly exposed to bacterial lawns and food-deprived conditions as above. Fifty worms were then collected into M9 containing 0.1 M sodium azide for paralysis, washed three times with M9 containing 1 mg/ml ampicillin (Biosharp) and 1 mg/ml gentamicin (Biosharp), and incubated for 30 min in antibiotic M9 to remove external bacteria. Worms were then lysed with a motorized pestle, serially diluted, and spread on the appropriate agar plates for each bacterial species. Plates were incubated overnight at 37 °C to determine CFUs.

Data analysis
Fluorescence, calcium, and pHluorin imaging data were analyzed using ImageJ (FIJI, version 1.50i). For neuronal imaging, individual cells were designated as separate regions of interest (ROIs). For the CRISPR knock-in SRI-36::mNeonGreen imaging, the dendritic region was selected as the ROI. For pHluorin imaging, the axonal region in the nerve ring was selected as the ROI. For intestinal fluorescence analysis, a single worm was assigned as the ROI. Calcium and pHluorin traces were first plotted as absolute fluorescence intensity (F) over time and then converted to bleach-corrected ΔF/F0 values using a custom MATLAB module94.

Statistical analyses and data visualization were performed using Origin 2019 (OriginLab). All diagram images were created using Microsoft PowerPoint 2024. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test for multiple group comparisons and two-sided unpaired t-test for two-group comparisons. Detailed statistical methods, sample sizes (n), and p-values are provided in the figure legends. Statistical significance was set at p < 0.05. No data were excluded from analysis, and all data are shown as mean ± SEM.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Lei et al. present a study that examines the role of gut-to-brain signaling in the simple animal C. elegans in modulating decision-making and behavioral responses to bacterial food cues and pathogenicity. The manuscript gets off to a strong start. The authors set up a nice assay that assesses the “risk-taking” behavior in crossing a noxious chemical barrier to access a food signal. The authors then examine how prior exposure to nutritious bacteria as well as three pathogenic bacteria, and also the effect of bacterial food removal, influence this decision making. The authors carry out a number of solid control experiments and confirm that fasting has a marked effect on subsequent behavioral responses. The authors find that exposure to pathogenic Pseudomonas aeruginosa reduces the animal’s risk-taking food-seeking behavior. Genetic experiments with tph-1 suggest a role for TPH-1 and the ADF neurons in mediating this response. The authors then conduct calcium-imaging experiments of ADF responses to show how ADF activity is influenced during conditions of fasting and Pseudomonas exposure. The authors use a candidate expression approach to identify a putative receptor, SRI-36, that is strongly induced in the ADF neurons by fasting and even more so by Pseudomonas infection. The authors show that ADF responses are depending on AAK-2, DAF-16, and the NSY-1-SEK-1 p38 pathway. Up until this point, the manuscript is quite impressive with a combination of beautiful genetics and imaging.\n\nHowever, once the manuscript begins to address site-of-action issues, there are serious concerns with the underlying method being used. The authors claim to be carrying out “cell-specific RNAi.” Methods are somewhat cursory, but it appears that the primary method being utilized is to use cell- or tissue-specific promoters to express + and – strands of a gene of interest off of a plasmid that has been introduced into cells of interest by the generation of transgenic strains. What is not taken into account is that this method does not take into account the ability for dsRNA to spread from cell-to-cell. For this reason, cell-specific RNAi methods are generally performed in an RNAi-defective strain with cell-specific rescue of RNAi (e.g., rde-1). The VP303 strain used for the screen for intestine-specific insulins has these attributes. But none of the other ostensibly cell-specific RNAi experiments utilize this strain or have any indication of a cell-specific rescue of RNAi activity. Thus, one cannot conclude anything about cell necessity from these site-of-action experiments. (It appears that the author used this method in a prior 2020 Nature Communications article as well.)\n\nUnless there is a trivial explanation that I am missing, this basic experimental oversight seriously undermines the confidence of this reviewer in the basic methodology and rigor of the manuscript. This is especially disappointing as the first part of the manuscript is remarkably impressive.", "reviewer_2": "“Intestinal pathogens override hunger-driven decision-making via immuneregulation of central serotonin signaling in C. elegans”\n\nSerotonin is known to react to various environmental stresses and internal states to modulate neural functions and generate behavioral responses. Some of these behavioral responses are orthogonal when the conditions are presented alone to the animals. How serotonin signaling regulates neural activity to produce coherent and balanced behavioral decisions under conditions demanding conflicting outputs from the nervous system is a fundamentally important but challenging question. In the above manuscript, Lei and Chen et al. set out to address this issue.\n\nThe authors employed a risk-taking decision paradigm in C. elegans where the worms’ attraction to an attractive odorant diacetyl (sensed by AWA sensory neuron) is hindered by a hyperosmotic glycerol barrier (sensed by ASH sensory neuron). They characterized how serotonin signaling modulated this decision-making process under two well-controlled conditions. While starvation promotes risk-taking as previously shown, the authors found that feeding on a pathogenic bacteria Pseudomonas aeruginosa PA14 suppressed the effect of starvation and inhibited the decision to cross the hyperosmotic barrier.\n\nUsing this assay together with cell-specific manipulation of serotonin production and neural activity, the authors showed that starvation and P. aeruginosa infection converged on the serotonin signal produced by ADF neurons to regulate behavior decision on diacetyl/glycerol response, and that starvation and infection both induced ADF response to diacetyl. Using previously published expression profiles, RNAi, and calcium imaging, CRISPR and cell-specific rescue, the authors further showed that starvation and P. aeruginosa infection upregulated the expression of a GPCR sri-36 in ADF and that sri-36 mediated diacetyl responses in fasted and infected worm in ADF neurons and in behavioral decision. The authors further identified two intestinal pathways, the aak-2/daf-16 pathway and pmk-2/daf-16 pathway, that upregulated sri-36 under fasting and infection conditions. These pathways upregulated expression of two insulin-like peptides, ins-37 and ins-7, in fasted and infected worms to modulate ADF expression of sri-36 and the behavioral decision between diacetyl/glycerol.\n\nFurthermore, the authors mapped the downstream neural circuit and serotonin receptors that mediated starvation and infection-regulated diacetyl response in ADF and behavioral decision, and demonstrated that a stronger release of serotonin from ADF in infected worms compared with starved worms suppressed the risk-taking effect of starvation. Based on these findings, they delineate a molecular strategy in a neuronal circuit that integrates food availability and infection to produce a balanced behavior decision between safety-seeking or risk-taking. The authors fully leverage the experimental power of C. elegans by taking multiple approaches to address the question at molecular, cellular and circuit levels. The findings are in general compelling. I have a few suggestions that will help complete the analysis.\n\n1. Is sri-36 expressed in any neurons other than ADF? The images in Figures 3g and 4d, 4f are too dark to see clearly. The authors should provide a more complete presentation on Psri-36::GFP expression.\n2. The GCaMP6 responses in ADF are relatively small in amplitude – do the transgenic worms expressing GCaMP6 in ADF neuron display normal behavioral decisions in the diacetyl/glycerol paradigm? Also importantly, the authors need to provide their methods for calcium imaging, one of the major experimental approaches in this paper.\n3. The authors need to document their source of P. aeruginosa PA14 strain.\n4. In Figure 5d, it should be ins-7 rescue, not inx-7 rescue.", "reviewer_3": "The authors show that fasting and intestinal infection with Pseudomonas aeruginosa PA14 produce opposite decisions in a C. elegans multisensory conflict assay by acting through distinct gut–brain endocrine pathways that converge on the same sensory neurons. Fasting engages intestinal AAK-2/DAF-16 to induce INS-37, whereas PA14 activates intestinal PMK-2 to induce INS-7; both act on ADF neurons via DAF-2 to upregulate the chemoreceptor SRI-36, but bias serotonin output toward approach or avoidance. The authors have an present impressive amount of work that defines a novel gut-brain communication pathway that dynamic sensory receptor tuning and divergent behavioral outcomes, offering a clear framework for how internal state reshapes sensory processing and decision-making. The depth of analysis is impressive including, identification chemoreceptor, Ca2+ imaging data and network analysis. The manuscript is clearly written and presents a compelling and conceptually novel gut–brain–behavior axis. I only have minor comments that could be clarified.\n\n1. Other pathogens (S. aureus and EPEC) have no effect (Fig. 1b):\nThe mechanistic basis for this difference is unclear. Do these strains fail to colonize or persist after fasting, or do they not activate PMK-2 signaling? Including colonization and persistence data after fasting, and/or PMK-2 activation levels, would clarify this point. If feasible, testing additional pathogenic bacteria that colonize and persist after fasting and induce PMK-2 immune signaling, and assessing whether they also reduce hunger-driven risk-taking, would further strengthen the argument.\n2. Colonization vs pathogenicity:\nCould it be that persistent gut bacteria could serve as internal “food-present” cues that suppress hunger responses? It would be informative to test a nonpathogenic strain that persists after fasting but does not activate PMK-2, to see if it fails to suppress escape.\n3. PA14 under well-fed conditions (Supplementary Fig. 1b):\nThe authors report that PA14 does not alter decision-making in well-fed animals, but it is possible that PA14 still induces PMK-2 immune signaling under these conditions. If so, the model might predict some reduction in risk-taking under a broader range of assay conditions, such as lower glycerol or higher diacetyl concentrations? For Fig. S1d and S1e, including well-fed controls for both OP50 and PA14 would strengthen interpretation. Fasting is known to increase diacetyl chemotaxis and fructose avoidance (ref. 6), yet these effects are not apparent here for glycerol avoidance. Testing a broader range of diacetyl and glycerol concentrations could help reveal subtle effects.\n5. 4 h vs 24 h infection (Supplementary Fig. 1g) and peptone supplementation (Fig. 1d):\nSimilar escape suppression seen after 4 h and 24 h PA14 exposure (S1g) is intriguing, as longer exposure might be expected to increase colonization and PMK-2 activation. Comparing colonization load and PMK-2 activation at both time points could clarify this. Likewise, while 0.35% peptone supplementation increases escape suppression, the effect is modest and may be influenced by outliers in the 0.25% condition.\n6. Residual fasting effect without 5-HT or ADF:\nEven with tph-1 loss (Fig. 1h), ADF silencing (Fig. 1j), or sri-36 deletion (Fig. 3f), fasting-induced escape remains higher than in well-fed animals. Including well-fed controls for both OP50 and PA14 in these experiments would help determine whether fasting still increases escape in these deficient backgrounds, suggesting the presence of parallel pathways.\n7. mod-5(n822) mutant phenotype:\nmod-5(n822) mutants, which have elevated extracellular 5-HT, show exaggerated fasting-induced escape (Fig. 1i). This seems inconsistent with the model, which predicts that high 5-HT should suppress escape in fasted worms, as chemogenetic activation of ADF reduces escape (Fig. 6h). Can the authors clarify\n8. Exogenous 5-HT tests:\nIt could be informative to test exogenous 5-HT supplementation in WT and tph-1 mutants under well-fed conditions, to see if low doses mimic fasting (increased escape) and high doses mimic PA14 infection (suppressed escape). Assessing effects on sri-36 expression, ADF activity, and diacetyl sensitivity would connect pharmacological effects to the proposed pathway.\n9. sri-36 expression levels:\nThe “moderate vs high” SRI-36 expression model could be strengthened by expressing sri-36 in ADF at moderate and high levels in sri-36 mutants, then measuring ADF diacetyl sensitivity, 5-HT release, and risk-taking. This would test whether moderate expression reproduces fasting effects and high expression reproduces PA14 effects, strengthening the link between receptor expression and behavioral tuning.\n10. ILP release mutants:\nhid-1 and egl-3 loss abolish ADF responses (Fig. 5a), consistent with the model that intestinal ILPs act via ADF DAF-2, unc-31(e169) mutants show no effect on ADF responses in any state (Fig. 2c). Does this mean that unc-31 is not requires for INS-37/7 release form the intestine?\nOther minor\n11. Supplementary Fig. 2 is not referenced in the main text.\n12 . line 295 The authors shoud weaken the statement : “These results demonstrate that hunger and P. aeruginosa infection induce intestinal INS-37 and INS-7 release, respectively” to indicate are consistent with.\n13. Different bacterial strains were cultured on distinct media and under different incubation conditions (e.g., NGM vs. peptone-supplemented NGM, tryptic soy agar, modified NGM with tryptophan; varied incubation times and temperatures). It would be helpful if the authors could briefly explain the rationale for these differences—for example, whether they are required for optimal bacterial growth, virulence factor expression, or compatibility with the assay conditions.\n14.For exogenous 5-HT stimulation, the authors applied serotonin directly to exposed neuronal somas via pressure ejection after incision. This is technically challenging. It would be useful to explain why this approach in more detail amd why this was chosen instead of exposing 5-HT ann intact animals during imaging. It would help readers understand the methodological rationale.\n15. For Fig. S1f, fasting typically increases locomotion speed and roaming, which is not observed here? It would be helpful to explain this difference. For Fig. S1f, fasting is generally associated with increased locomotion speed, which is not observed under the current assay conditions. It would be helpful if the authors could explain why they chose this specific setup (30 s acclimation followed by 1 min recording at 15 fps) and whether these parameters might limit detection of fasting-induced locomotion changes.\n16. Supplementary Fig. 2 is not referenced in the main text.", "reviewer_4": "In this submitted manuscript, Lei, Chen et al examine how C. elegans behavioral decisions are modulated by internal state. They use a classic paradigm where worms need to cross an aversive barrier to get to a chemoattractant Diacetyl. The authors first show that fasted animals cross the barrier more frequently, whereas P. aeruginosa-infected animals that are fasted do not. They find that these behavioral changes are dependent on serotonin production by ADF neurons. ADF is shown to directly respond to diacetyl, scaling up its response in fasted animals and even further upon infection. The authors then identify the sri-36 chemoreceptor as an ADF chemoreceptor that is upregulated upon fasting and even further upon infection. They show that the fasting effects work through AAK-2 and Insulin signaling, whereas the infection effects work through PMK/NSY/SEK signaling. Finally, the authors show that the fasting effects require the interneurons AIB/AUA/URX, whereas the infection effects require AIA/AIY. Serotonin receptors in these interneurons are identified.\n\nMost of the analyses in this paper are well done. I appreciated the identification of SRI-36 as an induced chemoreceptor and the important role of ADF in this behavior. However, I have two main reservations about publication of this study in Nature Communications:\n\n1) I worry that the final conceptual model in the paper doesn’t quite make sense in the context of the field. First, the authors argue for a biphasic curve where ADF promotes escape when it releases a medium amount of serotonin, but not when it is inactive or releasing a lot of serotonin. It’s just not clear how this could work. How would these subtly different levels of release result in vastly different behavioral responses? Showing how this works would require measuring dose-dependent serotonin responses in downstream interneurons much more carefully. Perhaps quantifying speed/turning and other parameters across these 3 internal states would help also. Moreover, the involvement of the interneurons doesn’t seem congruent with a substantial amount of prior knowledge in the field. The authors argue that AIB promotes escape behavior, but all prior work suggests that AIB enhances reversals (which would lead to a naïve guess that it inhibits escape behavior). The authors also argue that AIA/AIY promote staying behavior, but all prior work suggests these neurons drive forward movement (which would lead to a naïve guess that they promote escape). Overall, I worry that this ‘neural circuit mechanism’ part of the paper is underdeveloped and may not be correctly worked out yet.\n\n2) Regarding the first half of the paper – I find these results convincing and I appreciate the work that the authors put into this. But this is very well traveled ground. The idea that chemoreceptors are induced by fasting and infection has been established. The idea that Insulin signaling is involved in this process is also very well established. Relevant citations include: van der Linden et al 2007, Kyani-Rogers et al, 2022, Mclachlan et al, 2022, Wu et al, 2023, to name a few.\n\nI also have a few other minor comments:\n\n1. For the sri-36 gene expression studies, it would be preferable to use a CRISPR knock-in reporter, which has become the standard state-of-the-art in the field for these types of studies.\n2. Is pmk/sek signaling involved in the fasting effects? And is aak-2 involved in the infection effects? (or are these pathways totally separate and specific?)\n3. ADF releases other neurotransmitters – are they involved in these behaviors?"}, "author_response": "Response to Reviewers’ Comments\nWe thank the reviewers for their thorough evaluation of our manuscript and for their\nconstructive feedback. We have carefully addressed each point, performed additional\nexperiments, and made substantial changes to strengthen the study. Below, we\nprovide point-by-point responses. For clarity, reviewer comments are reproduced in\nitalics, followed by our responses, with reference to the relevant text, figure numbers,\nand references in the revised manuscript. All corresponding changes have been\nincorporated into the revised version and are highlighted in blue.\nEVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nLei et al. present a study that examines the role of gut-to-brain signaling in the simple\nanimal C. elegans in modulating decision-making and behavioral responses to\nbacterial food cues and pathogenicity. The manuscript gets off to a strong start. The\nauthors set up a nice assay that assesses the “risk-taking” behavior in crossing a\nnoxious chemical barrier to access a food signal. The authors then examine how\nprior exposure to nutritious bacteria as well as three pathogenic bacteria, and also\nthe effect of bacterial food removal, influence this decision making. The authors carry\nout a number of solid control experiments and confirm that fasting has a marked\neffect on subsequent behavioral responses. The authors find that exposure to\npathogenic Pseudomonas aeruginosa reduces the animal’s risk-taking food-seeking\nbehavior. Genetic experiments with tph-1 suggest a role for TPH-1 and the ADF\nneurons in mediating this response. The authors then conduct calcium-imaging\nexperiments of ADF responses to show how ADF activity is influenced during\nconditions of fasting and Pseudomonas exposure. The authors use a candidate\nexpression approach to identify a putative receptor, SRI-36, that is strongly induced\nin the ADF neurons by fasting and even more so by Pseudomonas infection. The\nauthors show that ADF responses are depending on AAK-2, DAF-16, and the NSY-1-\nSEK-1 p38 pathway. Up until this point, the manuscript is quite impressive with a\ncombination of beautiful genetics and imaging.\nResponse: We thank the reviewer for the overall positive assessment of our work.\nHowever, once the manuscript begins to address site-of-action issues, there are\nserious concerns with the underlying method being used. The authors claim to be\ncarrying out “cell-specific RNAi.” Methods are somewhat cursory, but it appears that\nthe primary method being utilized is to use cell- or tissue-specific promoters to\nexpress + and – strands of a gene of interest off of a plasmid that has been\nintroduced into cells of interest by the generation of transgenic strains. What is not\ntaken into account is that this method does not take into account the ability for\ndsRNA to spread from cell-to-cell. For this reason, cell-specific RNAi methods are\ngenerally performed in an RNAi-defective strain with cell-specific rescue of RNAi\n(e.g., rde-1). The VP303 strain used for the screen for intestine-specific insulins has\nthese attributes. But none of the other ostensibly cell-specific RNAi experiments\nutilize this strain or have any indication of a cell-specific rescue of RNAi activity.\nThus, one cannot conclude anything about cell necessity from these site-of-action\nexperiments. (It appears that the author used this method in a prior 2020 Nature\nCommunications article as well.)\nUnless there is a trivial explanation that I am missing, this basic experimental\noversight seriously undermines the confidence of this reviewer in the basic\nmethodology and rigor of the manuscript. This is especially disappointing as the first\npart of the manuscript is remarkably impressive.\nResponse: We thank the reviewer for raising this important methodological concern.\nIn our study, we employed a well-established tissue-specific RNAi strategy by co-\ninjecting plasmids encoding complementary sense and antisense fragments of the\ntarget gene under cell-specific promoters. This approach has been widely used in C.\nelegans and is considered reliable because, except for a few phasmid and male ray\nneurons, most neurons are intrinsically resistant to systemic RNAi owing to the\nabsence of the dsRNA transporter SID-1, which mediates inter-tissue RNAi\nspreading (Winston, W. M., Molodowitch, C. & Hunter, C. P., Science, 2002).\nConsistent with this, our previous work demonstrated that AVA-specific RNAi did not\nspread to A-type motor neurons, and vice versa (Liu et al., Nat Commun, 2017),\nvalidating the spatial restriction of this method.\nBeyond RNAi, our conclusions are supported by multiple orthogonal lines of\nevidence, including analyses of loss-of-function mutants and cell-specific rescues\n(sri-36, aak-2, ins-7, ser-4, lgc-50, and mod-1), as well as cell-specific expression of\nthe constitutively nuclear-localized DAF-16 (DAF-16aAM). These independent\napproaches consistently converge on the same mechanistic pathway, underscoring\nthe robustness of our conclusions.\nNevertheless, we recognize the reviewer’s point that potential off-target effects in\nIECs cannot be completely excluded. To rigorously address this concern, we\nrepeated the key intestine-specific RNAi experiments (aak-2, daf-16, nsy-1, sek-1,\npmk-2, hid-1, ins-37, and ins-7) in the systemic RNAi-defective sid-1(qt9) mutant\nbackground, in which dsRNA spreading is abolished (Winston, W. M., Molodowitch,\nC. & Hunter, C. P., Science, 2002). The results (revised Fig. 4a-g and Fig. 5a,c,d,f,g)\nare consistent with our original findings, confirming that the observed effects reflect\nintestine-specific knockdown. These findings are now described in the Results and\nclarified in the revised Methods as follows: “To confirm tissue specificity and exclude\nsystemic RNAi effects, intestine-specific RNAi experiments (aak-2, daf-16, nsy-1,\nsek-1, pmk-2, hid-1,ins-37, and ins-7) were repeated in the systemic RNAi-defective\nsid-1(qt9) mutant background, in which dsRNA spreading is abolished.”\nReviewer #2 (Remarks to the Author):\n“Intestinal pathogens override hunger-driven decision-making via immuneregulation\nof central serotonin signaling in C. elegans”\nSerotonin is known to react to various environmental stresses and internal states to\nmodulate neural functions and generate behavioral responses. Some of these\nbehavioral responses are orthogonal when the conditions are presented alone to the\nanimals. How serotonin signaling regulates neural activity to produce coherent and\nbalanced behavioral decisions under conditions demanding conflicting outputs from\nthe nervous system is a fundamentally important but challenging question. In the\nabove manuscript, Lei and Chen et al. set out to address this issue.\nThe authors employed a risk-taking decision paradigm in C. elegans where the\nworms’ attraction to an attractive odorant diacetyl (sensed by AWA sensory neuron)\nis hindered by a hyperosmotic glycerol barrier (sensed by ASH sensory neuron).\nThey characterized how serotonin signaling modulated this decision-making process\nunder two well-controlled conditions. While starvation promotes risk-taking as\npreviously shown, the authors found that feeding on a pathogenic bacteria\nPseudomonas aeruginosa PA14 suppressed the effect of starvation and inhibited the\ndecision to cross the hyperosmotic barrier.\nUsing this assay together with cell-specific manipulation of serotonin production and\nneural activity, the authors showed that starvation and P. aeruginosa infection\nconverged on the serotonin signal produced by ADF neurons to regulate behavior\ndecision on diacetyl/glycerol response, and that starvation and infection both induced\nADF response to diacetyl. Using previously published expression profiles, RNAi, and\ncalcium imaging, CRISPR and cell-specific rescue, the authors further showed that\nstarvation and P. aeruginosa infection upregulated the expression of a GPCR sri-36\nin ADF and that sri-36 mediated diacetyl responses in fasted and infected worm in\nADF neurons and in behavioral decision. The authors further identified two intestinal\npathways, the aak-2/daf-16 pathway and pmk-2/daf-16 pathway, that upregulated sri-\n36 under fasting and infection conditions. These pathways upregulated expression of\ntwo insulin-like peptides, ins-37 and ins-7, in fasted and infected worms to modulate\nADF expression of sri-36 and the behavioral decision between diacetyl/glycerol.\nFurthermore, the authors mapped the downstream neural circuit and serotonin\nreceptors that mediated starvation and infection-regulated diacetyl response in ADF\nand behavioral decision, and demonstrated that a stronger release of serotonin from\nADF in infected worms compared with starved worms suppressed the risk-taking\neffect of starvation. Based on these findings, they delineate a molecular strategy in a\nneuronal circuit that integrates food availability and infection to produce a balanced\nbehavior decision between safety-seeking or risk-taking. The authors fully leverage\nthe experimental power of C. elegans by taking multiple approaches to address the\nquestion at molecular, cellular and circuit levels. The findings are in general\ncompelling. I have a few suggestions that will help complete the analysis.\nResponse: We thank the reviewer for the overall positive assessment of our work.\n1. Is sri-36 expressed in any neurons other than ADF? The images in Figures 3g and\n4d, 4f are too dark to see clearly. The authors should provide a more complete\npresentation on Psri-36::GFP expression.\nResponse: We thank the reviewer for raising this important point. We have improved\nPsri-36::GFP images and now included a whole-body view. The data show that sri-36\nexpression is restricted to ADF neurons during P. aeruginosa infection, with no\ndetectable expression in other neurons (new Supplementary Fig. 3b). To further\nvalidate this observation, we generated a CRISPR knock-in sri-36::mNeonGreen\nreporter with mNeonGreen inserted at the C-terminus of the endogenous locus. This\nknock-in reporter corroborated the Psri-36::GFP expression pattern (new\nSupplementary Fig. 3c). These findings are now incorporated into the Results.\n2. The GCaMP6 responses in ADF are relatively small in amplitude – do the\ntransgenic worms expressing GCaMP6 in ADF neuron display normal behavioral\ndecisions in the diacetyl/glycerol paradigm? Also importantly, the authors need to\nprovide their methods for calcium imaging, one of the major experimental\napproaches in this paper.\nResponse: We agree that it is important to confirm that GCaMP6 expression in ADF\ndoes not interfere with behavior. We tested the transgenic worms expressing\nGCaMP6 in ADF neuron (xyhEx446[Psrh-142::HisCl1::SL2::GCaMP6s, Punc-\n122::GFP]) and found that they exhibited normal escape behavior compared to wild\ntype, indicating that GCaMP6 expression does not affect decision-making (new\nSupplementary Fig. 2e). We have also expanded the Methods to include a detailed\ndescription of the calcium imaging protocol (revised Methods, “Imaging”).\n3. The authors need to document their source of P. aeruginosa PA14 strain.\nResponse: We thank the reviewer for this suggestion. The bacterial strains used in\nthis study are now listed in Table 1 and were obtained from the following sources: E.\ncoli OP50 from the Caenorhabditis Genetics Center (CGC); P. aeruginosa PA14 and\nS. aureus NCTC8325 from Dr. Jianke Gong (Huazhong University of Science and\nTechnology); PA14-dsRed from Dr. Bin Qi (Yunnan University); EPEC and E. coli\nHT115 from Dr. Anbing Shi (Huazhong University of Science and Technology); and\nPA14-gacA(-) from Taihong Wu (Hunan University).\n4. In Figure 5d, it should be ins-7 rescue, not inx-7 rescue.\nResponse: Corrected, thanks.\nReviewer #3 (Remarks to the Author):\nThe authors show that fasting and intestinal infection with Pseudomonas aeruginosa\nPA14 produce opposite decisions in a C. elegans multisensory conflict assay by\nacting through distinct gut–brain endocrine pathways that converge on the same\nsensory neurons. Fasting engages intestinal AAK-2/DAF-16 to induce INS-37,\nwhereas PA14 activates intestinal PMK-2 to induce INS-7; both act on ADF neurons\nvia DAF-2 to upregulate the chemoreceptor SRI-36, but bias serotonin output toward\napproach or avoidance. The authors have an present impressive amount of work that\ndefines a novel gut-brain communication pathway that dynamic sensory receptor\ntuning and divergent behavioral outcomes, offering a clear framework for how\ninternal state reshapes sensory processing and decision-making. The depth of\nanalysis is impressive including, identification chemoreceptor, Ca2+ imaging data\nand network analysis. The manuscript is clearly written and presents a compelling\nand conceptually novel gut–brain–behavior axis. I only have minor comments that\ncould be clarified.\nResponse: We thank the reviewer for the overall positive assessment of our work.\n1. Other pathogens (S. aureus and EPEC) have no effect (Fig. 1b):\nThe mechanistic basis for this difference is unclear. Do these strains fail to colonize\nor persist after fasting, or do they not activate PMK-2 signaling? Including\ncolonization and persistence data after fasting, and/or PMK-2 activation levels, would\nclarify this point. If feasible, testing additional pathogenic bacteria that colonize and\npersist after fasting and induce PMK-2 immune signaling, and assessing whether\nthey also reduce hunger-driven risk-taking, would further strengthen the argument.\nResponse: We thank the reviewer for this insightful comment. We have now\nperformed colonization assays for S. aureus and EPEC after fasting, showing that S.\naureus colonized the intestine whereas EPEC did not, but neither pathogen induced\nPins-7::GFP expression (new Supplementary Fig. 5e,f). These results are consistent\nwith their lack of behavioral effect, indicating that activation of the intestinal PMK-2\nimmune signaling pathway is required to suppress hunger-driven risk-taking. These\nfindings are now incorporated into the Results and further discussed in the\nDiscussion. We agree that testing additional pathogens will be valuable to generalize\nthis mechanism and have emphasized this point in the revised Discussion as follows:\n“Future studies will be important to test additional pathogens that both colonize and\nactivate PMK-2 signaling, to determine whether this mechanism for overriding\nhunger-driven decision-making is broadly conserved across diverse microbial\ninfections.”\n2. Colonization vs pathogenicity:\nCould it be that persistent gut bacteria could serve as internal “food-present” cues\nthat suppress hunger responses? It would be informative to test a nonpathogenic\nstrain that persists after fasting but does not activate PMK-2, to see if it fails to\nsuppress escape.\nResponse: We thank the reviewer for this thoughtful comment. However, our results\nargue against this possibility and instead support that suppression of hunger\nresponses requires immune activation by P. aeruginosa:\n1. Pathogen specificity: Among the three pathogens tested, only P. aeruginosa\nsuppressed hunger-driven escape, whereas S. aureus colonized the intestine but\nhad no effect (Fig. 1b and new Supplementary Fig. 5e). Knockdown of nol-6, which\nreduces P. aeruginosa colonization, attenuated suppression (Fig. 1e,f). These results\nindicate that colonization is necessary but not sufficient.\n2. Virulence requirement: Suppression required live and virulent P. aeruginosa.\nNeither supernatant, odors, heat-killed cells, nor the nonpathogenic PA14-gacA(-)\nmutant suppressed escape (Fig. 1c). Conversely, enhancing virulence with 0.35%\npeptone further strengthened suppression (Fig. 1d). Thus, virulence rather than\npersistence alone is critical.\n3. Immune signaling dependence: Genetic analyses showed that suppression\ndepends on intestinal PMK-2/p38 MAPK signaling (Fig. 4), which is activated by P.\naeruginosa but not by S. aureus (new Supplementary Fig. 5f), establishing a\nmechanistic requirement for immune activation.\n4. Neural and sensory signatures: Infection produced distinct physiological and\nneural changes not observed with simple feeding. Specifically, SRI-36 expression in\nADF, diacetyl-evoked calcium responses in ADF and AWA, and glycerol-evoked\ncalcium responses in ASH all differed markedly between well-fed and P. aeruginosa-\ninfected worms, arguing against infection simply mimicking food presence.\n5. Nonpathogenic strain test: We examined the nonpathogenic P. aeruginosa mutant\nstrain PA14-gacA(-) and found that it colonized the intestine nearly 16-fold more than\nE. coli after fasting (CFUs: 555 ± 87.63 vs. 35.33 ± 10.64) (new Supplementary Fig.\n5e). However, PA14-gacA(-) did not induced Pins-7::GFP expression (new\nSupplementary Fig. 5f) and failed to suppress hunger-driven escape (Fig. 1c), in\ncontrast to wild-type P. aeruginosa.\n3. PA14 under well-fed conditions (Supplementary Fig. 1b):\nThe authors report that PA14 does not alter decision-making in well-fed animals, but\nit is possible that PA14 still induces PMK-2 immune signaling under these conditions.\nIf so, the model might predict some reduction in risk-taking under a broader range of\nassay conditions, such as lower glycerol or higher diacetyl concentrations? For Fig.\nS1d and S1e, including well-fed controls for both OP50 and PA14 would strengthen\ninterpretation. Fasting is known to increase diacetyl chemotaxis and fructose\navoidance (ref. 6), yet these effects are not apparent here for glycerol avoidance.\nTesting a broader range of diacetyl and glycerol concentrations could help reveal\nsubtle effects.\nResponse: We thank the reviewer for this insightful comment. As expected, our new\nexperiments showed that P. aeruginosa induced Pins-7::GFP expression in well-fed\nworms (new Supplementary Fig. 5g), indicating activation of PMK-2 signaling.\nConsistently, P. aeruginosa also induced Psri-36::GFP expression, but the signal was\nvery weak (new Supplementary Fig. 5h). To address the reviewer’s suggestion\nregarding behavioral effects:\n1. We added well-fed controls for both E. coli and P. aeruginosa in the diacetyl and\nglycerol assays (revised Supplementary Fig. 1d,e). As expected, fasting increased\ndiacetyl chemotaxis (revised Supplementary Fig. 1d). For glycerol avoidance in the\nabsence of diacetyl, fasting increased escape at 2 M glycerol, but not at 1 M (likely\ntoo weak) or 3 M (likely because the motivation to escape was outweighed by the\ninhibitory effect of this high glycerol concentration on locomotion) (revised\nSupplementary Fig. 1e). Nevertheless, there was no significant difference between E.\ncoli- and P. aeruginosa-fed worms under either well-fed or fasted conditions in both\nunisensory assays (revised Supplementary Fig. 1d,e). These findings are consistent\nwith previous results (Ghosh, D. D. et al. Neuron, 2016) and with our data showing\nthat fasting enhanced AWA diacetyl responses and reduced ASH glycerol responses,\nwhereas P. aeruginosa infection did not further alter their responses (Supplementary\nFig. 2b,c). Together, these results support our conclusion that P. aeruginosa does not\naffect unisensory decision-making.\n2. As suggested, we also tested additional glycerol concentrations (1 M and 2 M)\npaired with 1:1000 diacetyl in well-fed worms. Consistent with the results at 3 M\nglycerol (Supplementary Fig. 1b), P. aeruginosa infection did not affect escape under\nthese conditions (new Supplementary Fig. 5i), suggesting that immune activation has\nonly subtle effects on risk-taking in the absence of fasting.\nTogether, these results suggest that while P. aeruginosa activates intestinal immune\nsignaling in both well-fed and fasted worms, its behavioral suppression is most\npronounced under fasting, when hunger-driven risk-taking is elevated. These findings\nare now incorporated into the Results.\n5. 4 h vs 24 h infection (Supplementary Fig. 1g) and peptone supplementation (Fig.\n1d):\nSimilar escape suppression seen after 4 h and 24 h PA14 exposure (S1g) is\nintriguing, as longer exposure might be expected to increase colonization and PMK-2\nactivation. Comparing colonization load and PMK-2 activation at both time points\ncould clarify this. Likewise, while 0.35% peptone supplementation increases escape\nsuppression, the effect is modest and may be influenced by outliers in the 0.25%\ncondition.\nResponse: We thank the reviewer for this insightful comment. To address these\npoints, we quantified both intestinal PA14-dsRed colonization and Pins-7::GFP\nexpression after 4 h and 24 h of infection (new Supplementary Fig. 1i and new\nSupplementary Fig. 5a), and under 0.25% vs. 0.35% peptone supplementation (new\nSupplementary Fig. 1h and new Supplementary Fig. 5b).\n1. 4 h vs. 24 h infection: PA14-dsRed colonization and Pins-7::GFP expression did\nnot show significant difference, consistent with the similar escape behavior. These\nresults suggest that infection and immune activation reach a saturating level by 4 h.\n2. 0.25% vs. 0.35% peptone: Both PA14-dsRed colonization and Pins-7::GFP\nexpression were higher at 0.35% peptone, consistent with the stronger escape\nsuppression. These results indicate that enhanced virulence promotes colonization\nand immune activation, but the behavioral response is limited by ceiling effects.\nThese findings have been incorporated into the Results.\n6. Residual fasting effect without 5-HT or ADF:\nEven with tph-1 loss (Fig. 1h), ADF silencing (Fig. 1j), or sri-36 deletion (Fig. 3f),\nfasting-induced escape remains higher than in well-fed animals. Including well-fed\ncontrols for both OP50 and PA14 in these experiments would help determine whether\nfasting still increases escape in these deficient backgrounds, suggesting the\npresence of parallel pathways.\nResponse: We thank the reviewer for this insightful comment. We have now\nincluded well-fed controls for E. coli and P. aeruginosa in these deficient\nbackgrounds (new Supplementary Fig. 8). These data show that fasting still\nincreased escape even when 5-HT synthesis, ADF activity, or sri-36 was disrupted,\nindicating the involvement of 5-HT-independent, parallel mechanisms. We have\nexpanded the Discussion to note this point as follows: “Notably, fasting still increased\nescape even when 5-HT synthesis, ADF activity, or sri-36 was disrupted, suggesting\nthe presence of parallel, 5-HT-independent pathways. These may include other\nneuromodulators such as tyramine, dopamine, and additional signals that contribute\nto hunger-driven risk-taking. Future work will be needed to delineate these parallel\nmechanisms and determine how they interact with the 5-HT axis to shape adaptive\ndecision-making.”\n7. mod-5(n822) mutant phenotype:\nmod-5(n822) mutants, which have elevated extracellular 5-HT, show exaggerated\nfasting-induced escape (Fig. 1i). This seems inconsistent with the model, which\npredicts that high 5-HT should suppress escape in fasted worms, as chemogenetic\nactivation of ADF reduces escape (Fig. 6h). Can the authors clarify\nResponse: We agree that the mod-5(n822) phenotype may appear paradoxical at\nfirst glance. We propose that this difference reflects the spatial and temporal\ndynamics of 5-HT signaling. In mod-5(n822) mutants, defective reuptake elevates\nbasal extracellular 5-HT levels, producing a chronic and diffuse increase that likely\npotentiates hunger-driven escape by enhancing overall 5-HT tone. In contrast,\nchemogenetic activation of ADF induces a strong, acute, and spatially restricted 5-HT\nrelease at ADF synapses, which suppresses risk-taking. Thus, the mod-5(n822)\nphenotype is consistent with our model: moderate increases in 5-HT release\npromotes risk-taking, whereas excessive ADF-specific release suppresses it.\n8. Exogenous 5-HT tests:\nIt could be informative to test exogenous 5-HT supplementation in WT and tph-1\nmutants under well-fed conditions, to see if low doses mimic fasting (increased\nescape) and high doses mimic PA14 infection (suppressed escape). Assessing\neffects on sri-36 expression, ADF activity, and diacetyl sensitivity would connect\npharmacological effects to the proposed pathway.\nResponse: We thank the reviewer for this insightful suggestion. We did not pursue\nexogenous 5-HT supplementation in this study for two reasons. First, exogenous 5-\nHT broadly activates serotonergic targets throughout the animal, whereas our model\nfocuses on state-dependent modulation of ADF-derived 5-HT and its circuit-specific\neffects. Second, exogenous 5-HT can produce pleiotropic and dose-dependent\noutcomes that are difficult to interpret, as its effects vary with concentration, timing,\nand site of application. Instead, we directly tested our model using targeted genetic\nand physiological approaches by monitoring sri-36 expression, ADF activity, and ADF\n5-HT release (synapto-pHluorin imaging). These complementary strategies allowed\nus to causally link intestinal signaling, ADF chemoreceptor expression, and behavior.\n9. sri-36 expression levels:\nThe “moderate vs high” SRI-36 expression model could be strengthened by\nexpressing sri-36 in ADF at moderate and high levels in sri-36 mutants, then\nmeasuring ADF diacetyl sensitivity, 5-HT release, and risk-taking. This would test\nwhether moderate expression reproduces fasting effects and high expression\nreproduces PA14 effects, strengthening the link between receptor expression and\nbehavioral tuning.\nResponse: We thank the reviewer for this thoughtful suggestion. We previously\ninjected 25 ng/µl of the Psrh-142::sri-36 plasmid and observed rescue of both ADF\ndiacetyl responses and escape in fasted and infected worms (Fig. 3e,f). However, the\nexact expression levels could not be determined in the absence of a fluorescent\nmarker. To directly test the effect of expression level, we expressed SRI-36 at two\ndifferent levels in sri-36(plc921) mutants by injecting 5 ng/µl (“low” expression) or 75\nng/µl (“high” expression) of the Psrh-142::sri-36::mStrawberry plasmid. mCherry\nfluorescence intensity confirmed lower SRI-36 expression in ADF at 5 ng/µl\ncompared to 75 ng/µl (Supplementary Fig. 3d). In fasted worms, low-level expression\nrestored moderate ADF diacetyl responses, 5-HT release, and escape, resembling\nfasted controls. In contrast, high-level expression enhanced ADF diacetyl responses\nand 5-HT release, mimicking the suppression of escape observed during P.\naeruginosa infection (revised Fig. 3e,f and Fig. 6g). These results directly support\nour model, demonstrating that SRI-36 expression levels tune ADF sensitivity, 5-HT\noutput, and behavioral choice. The corresponding findings have been incorporated\ninto the Results.\n10. ILP release mutants:\nhid-1 and egl-3 loss abolish ADF responses (Fig. 5a), consistent with the model that\nintestinal ILPs act via ADF DAF-2, unc-31(e169) mutants show no effect on ADF\nresponses in any state (Fig. 2c). Does this mean that unc-31 is not requires for INS-\n37/7 release form the intestine?\nResponse: We thank the reviewer for this important point. Based on our results (Fig.\n2c), UNC-31 is not required for fasting-induced INS-37 release or P. aeruginosa-\ninduced INS-7 release from IECs under our experimental conditions. This is\nconsistent with published reports showing that UNC-31 is not expressed in the\nintestine (Speese, S. et al., J Neurosci, 2007; Cornell, R., Cao, W., Liu, J. & Pocock,\nR, J Neurosci, 2022).\nOther minor\n11. Supplementary Fig. 2 is not referenced in the main text.\nResponse: We thank the reviewer for pointing this out. This figure was inadvertently\nincluded from an earlier draft and is not part of the current study. We have removed it\nfrom the revised submission.\n12 . line 295 The authors shoud weaken the statement : “These results demonstrate\nthat hunger and P. aeruginosa infection induce intestinal INS-37 and INS-7 release,\nrespectively” to indicate are consistent with.\nResponse: We thank the reviewer for this helpful suggestion. We have revised the\nstatement to: “These results are consistent with a model in which hunger and P.\naeruginosa infection induce intestinal INS-37 and INS-7 release, respectively,”\n13. Different bacterial strains were cultured on distinct media and under different\nincubation conditions (e.g., NGM vs. peptone-supplemented NGM, tryptic soy agar,\nmodified NGM with tryptophan; varied incubation times and temperatures). It would\nbe helpful if the authors could briefly explain the rationale for these differences—for\nexample, whether they are required for optimal bacterial growth, virulence factor\nexpression, or compatibility with the assay conditions.\nResponse: We thank the reviewer for this insightful comment. Distinct culture\nconditions were selected to ensure optimal growth, virulence factor expression, and\ncompatibility with C. elegans assays (Wu, T. et al., Nature, 2023; Anyanful, A.,\nEasley, K. A., Benian, G. M. & Kalman, Cell Host Microbe, 2009). Specifically:\n1. P. aeruginosa was plated on NGM + 0.25% peptone, which supports robust\nvirulence factor production while maintaining animal viability for behavioral assays.\n0.35% peptone was used to further enhance virulence.\n2. S. aureus was cultured on tryptic soy agar with nalidixic acid, which both optimizes\ngrowth of this Gram-positive pathogen and prevents contamination.\n3. EPEC was grown on modified NGM containing tryptophan, a condition previously\nreported to enhance its virulence toward C. elegans.\nWe have added these explanations into the Methods (“Bacterial strains and culture”).\n14.For exogenous 5-HT stimulation, the authors applied serotonin directly to exposed\nneuronal somas via pressure ejection after incision. This is technically challenging. It\nwould be useful to explain why this approach in more detail amd why this was\nchosen instead of exposing 5-HT ann intact animals during imaging. It would help\nreaders understand the methodological rationale.\nResponse: We thank the reviewer for this thoughtful comment. The targeted\ninterneurons (AIA, AIY, AIB, and AUA) are located deep within the head and are not\ndirectly accessible to exogenously applied 5-HT in intact animals. Therefore,\nexposing intact worms to 5-HT would not allow precise or reproducible stimulation of\nthese neurons. To overcome this limitation, we used an established approach (Zhan,\nX. et al., Nat Commun, 2023), in which immobilized animals are incised along the\nglued region to expose neuron somas. 5-HT was then directly applied onto the soma\nby pressure ejection, enabling spatially precise, dose-controlled, and reproducible\nstimulation. We have clarified this rationale in the revised Methods (“Imaging”).\n15. For Fig. S1f, fasting typically increases locomotion speed and roaming, which is\nnot observed here? It would be helpful to explain this difference. For Fig. S1f, fasting\nis generally associated with increased locomotion speed, which is not observed\nunder the current assay conditions. It would be helpful if the authors could explain\nwhy they chose this specific setup (30 s acclimation followed by 1 min recording at\n15 fps) and whether these parameters might limit detection of fasting-induced\nlocomotion changes.\nResponse: We thank the reviewer for raising this important point. Although fasting is\noften thought to increase locomotion or roaming, published evidence indicates that\nthis effect is context-dependent. For example, Sawin et al. (Neuron, 2000) reported\nno significant difference in locomotory rate (body bends) between 30 min-fasted and\nwell-fed animals, consistent with our results obtained using the Track-A-Worm\nautomated tracking system. Regarding our recording setup, we used a 30 s\nacclimation and 1 min recording at 15 fps to minimize environmental variability and\nensure reproducibility across groups. This protocol provides robust sampling of basal\nlocomotion aligned with our behavioral paradigms. While longer recordings might\nreveal subtle differences, both our data and Sawin et al. suggest that fasting does not\nmarkedly alter basal locomotion under these conditions.\n16. Supplementary Fig. 2 is not referenced in the main text.\nResponse: We thank the reviewer for pointing this out. As noted in our response to\ncomment 11, this figure was inadvertently included from an earlier draft and has been\nremoved from the revised manuscript.\nReviewer #4 (Remarks to the Author):\nIn this submitted manuscript, Lei, Chen et al examine how C. elegans behavioral\ndecisions are modulated by internal state. They use a classic paradigm where worms\nneed to cross an aversive barrier to get to a chemoattractant Diacetyl. The authors\nfirst show that fasted animals cross the barrier more frequently, whereas P.\naeruginosa-infected animals that are fasted do not. They find that these behavioral\nchanges are dependent on serotonin production by ADF neurons. ADF is shown to\ndirectly respond to diacetyl, scaling up its response in fasted animals and even\nfurther upon infection. The authors then identify the sri-36 chemoreceptor as an ADF\nchemoreceptor that is upregulated upon fasting and even further upon infection. They\nshow that the fasting effects work through AAK-2 and Insulin signaling, whereas the\ninfection effects work through PMK/NSY/SEK signaling. Finally, the authors show\nthat the fasting effects require the interneurons AIB/AUA/URX, whereas the infection\neffects require AIA/AIY. Serotonin receptors in these interneurons are identified.\nMost of the analyses in this paper are well done. I appreciated the identification of\nSRI-36 as an induced chemoreceptor and the important role of ADF in this behavior.\nHowever, I have two main reservations about publication of this study in Nature\nCommunications:\nResponse: We thank the reviewer for the overall positive assessment of our work.\n1) I worry that the final conceptual model in the paper doesn’t quite make sense in\nthe context of the field. First, the authors argue for a biphasic curve where ADF\npromotes escape when it releases a medium amount of serotonin, but not when it is\ninactive or releasing a lot of serotonin. It’s just not clear how this could work. How\nwould these subtly different levels of release result in vastly different behavioral\nresponses? Showing how this works would require measuring dose-dependent\nserotonin responses in downstream interneurons much more carefully. Perhaps\nquantifying speed/turning and other parameters across these 3 internal states would\nhelp also. Moreover, the involvement of the interneurons doesn’t seem congruent\nwith a substantial amount of prior knowledge in the field. The authors argue that AIB\npromotes escape behavior, but all prior work suggests that AIB enhances reversals\n(which would lead to a naïve guess that it inhibits escape behavior). The authors also\nargue that AIA/AIY promote staying behavior, but all prior work suggests these\nneurons drive forward movement (which would lead to a naïve guess that they\npromote escape). Overall, I worry that this ‘neural circuit mechanism’ part of the\npaper is underdeveloped and may not be correctly worked out yet.\nResponse: We thank the reviewer for this perceptive and constructive critique. Our\ndata support a model in which moderate ADF activation (as during fasting) promotes\nrisk-taking, whereas strong ADF activation (as during infection) suppresses it. We\ninterpret this as a level-dependent mechanism: moderate 5-HT release preferentially\nengages the ADF-AIB/AUA pathway, while excessive release recruits the ADF-\nAIA/AIY pathway, producing opposing behavioral outcomes. Mechanistically, distinct\n5-HT receptors and target neurons likely have different activation thresholds and\nfunctional responses, allowing graded 5-HT release to differentially recruit\ndownstream circuits. This logic is consistent with whole-brain analyses showing that\n5-HT modulation is highly site- and receptor-specific, with different 5-HT\nconcentrations and release patterns engaging distinct network modules (Dag, U. et\nal., Cell, 2023; Flavell, Steven W. et al., Cell, 2013). We have clarified this conceptual\nframework in the Discussion and now noted that “Future work should quantify 5-HT\ndose-response effects on downstream interneurons (e.g., changes in speed or\nturning dynamics across internal states) to further refine this circuit model.”\nRegarding interneuron assignments, we agree with the reviewer’s summary of prior\nliterature and clarify that our model aligns with established neuronal functions\n(Metaxakis, A., Petratou, D. & Tavernarakis, N., Open Biol, 2018; Ezcurra, M. et al., J\nNeurosci, 2016):\n1. AIB promotes avoidance; thus, its inhibition by moderate ADF activity reduces\navoidance, facilitating risk-taking.\n2. AUA suppresses avoidance and biases animals toward approach; its activation by\nmoderate ADF activity promotes escape across the barrier.\n3. AIA and AIY promote attraction and forward locomotion; under infection, strong\nADF activation suppresses AIA/AIY, thereby reducing food approach.\nTogether, these results suggest that ADF flexibly recruits distinct interneuron modules\nto implement opposing behavioral strategies according to metabolic and immune\nstates. Our imaging, chemogenetic, and behavioral data consistently support this\nframework.\n2) Regarding the first half of the paper – I find these results convincing and I\nappreciate the work that the authors put into this. But this is very well traveled\nground. The idea that chemoreceptors are induced by fasting and infection has been\nestablished. The idea that Insulin signaling is involved in this process is also very well\nestablished. Relevant citations include: van der Linden et al 2007, Kyani-Rogers et\nal, 2022, Mclachlan et al, 2022, Wu et al, 2023, to name a few.\nResponse: We thank the reviewer for acknowledging the strength of the study and\nfor highlighting relevant prior work. We agree that chemoreceptor regulation by\ninternal states and the involvement of insulin signaling have been elegantly\nestablished in earlier studies (van der Linden, A. M., Nolan, K. M. & Sengupta, P.,\nEMBO J, 2007; Kyani-Rogers, T. et al., Genetics, 2022; McLachlan, I. G. et al., eLife,\n2022; Wu, T. et al., Nature, 2023). These studies showed that fasting, infection, or\ndevelopmental history can modulate chemoreceptor expression through pathways\ninvolving DAF-16/FOXO, MEF2, or ZIP-5, thereby influencing sensory and behavioral\nresponses. Our work substantially extends these concepts in several key ways:\n1. Identification of SRI-36 as a diacetyl receptor or essential subunit: We\ndemonstrate that SRI-36 is both necessary and sufficient to tune ADF diacetyl\nsensitivity and risk-taking behavior. To our knowledge, SRI-36 is the first\nchemoreceptor shown to be induced by both metabolic (fasting) and immune\n(pathogen) signaling in C. elegans.\n2. Integration of immune and metabolic signaling via a FOXO-to-FOXO axis: We\ndelineate how two distinct intestinal pathways, AAK-2-DAF-16 during fasting and\nPMK-2-DAF-16 during infection, converge on ADF, representing a previously\nunrecognized form of immune-metabolic integration for gut-to-brain communication.\n3. Linking receptor tuning to circuit and behavioral outcomes: Beyond describing\nchemoreceptor regulation, we establish the causal pathway from intestinal signaling,\nthrough SRI-36 induction and ADF 5-HT release, to downstream interneurons that\nimplement opposing behavioral strategies. This circuit-level framework connects\nintestinal signaling to defined neuronal pathways, substantially extending prior\nobservations.\n4. State-specific signaling dependence: Our findings reveal a gut-to-brain mechanism\nwhereby hunger promotes risk-taking while infection suppresses it, both by\nmodulating central 5-HT signaling through distinct intestinal pathways. This provides\nmechanistic insight into how internal states reshape decision-making.\nTogether, our findings build on and go beyond previous work. We have cited these\nrelevant studies in the revised Discussion.\nI also have a few other minor comments:\n1. For the sri-36 gene expression studies, it would be preferable to use a CRISPR\nknock-in reporter, which has become the standard state-of-the-art in the field for\nthese types of studies.\nResponse: We thank the reviewer for this valuable suggestion. As suggested, we\ngenerated a CRISPR knock-in sri-36::mNeonGreen reporter, with mNeonGreen\ninserted at the C-terminus of the endogenous locus. This reporter confirmed that\nSRI-36 expression is specifically induced in ADF neurons upon fasting, further\nenhanced by P. aeruginosa infection, and localized to the sensory cilia (new\nSupplementary Fig. 3c). These results validate our original conclusions and support\nthe role of SRI-36 in detecting environmental odors. The new data have been\nincorporated into the Results.\n2. Is pmk/sek signaling involved in the fasting effects? And is aak-2 involved in the\ninfection effects? (or are these pathways totally separate and specific?)\nResponse: We thank the reviewer for this insightful question. To address it, we\nperformed reciprocal reporter assays. We found that fasting-induced Pins-37::GFP\nexpression was not altered by intestinal pmk-2 RNAi (new Supplementary Fig. 5c). In\ncontrast, P. aeruginosa infection-induced Pins-7::GFP expression was further\nenhanced in aak-2 RNAi worms after 1 h and 2 h of infection, but not at 4 h,\nsuggesting a transient peak of infection-induced ins-7 expression (new\nSupplementary Fig. 5d). These results indicate that pmk-2 signaling is not required\nfor fasting-induced ins-37 expression, whereas aak-2 normally acts to dampen ins-7\ninduction during infection. Thus, while aak-2 and pmk-2 serve as primary mediators\nof fasting- and infection-induced responses, respectively, our findings also reveal\nlimited cross-talk between these pathways. These new data have been incorporated\ninto the Results.\n3. ADF releases other neurotransmitters – are they involved in these behaviors?\nResponse: We thank the reviewer for this insightful question. Based on current\nknowledge, ADF is also cholinergic (Pereira, L. et al., eLife, 2015). To test whether\nacetylcholine (ACh) contributes, we performed ADF-specific unc-17 RNAi to reduce\nvesicular ACh release. This manipulation did not significantly affect escape in either\nthe fasted or infected state (new Supplementary Fig. 1j). These findings indicate that\nACh release from ADF is not required for the state-dependent regulation of decision-\nmaking, although it may contribute to other ADF-mediated functions. These new data\nhave been incorporated into the Results."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript by Liu and colleagues does not address the principal concern raised in my initial review. The authors do not seem to grasp the seriousness of their lack of understanding of their own experimental system.\n\nThe authors state that their method is one that is widely accepted. In fact, the issue of whether dsRNA can spread from neurons has been examined by Jose and Hunter and colleagues. Specifically, as noted by Ravikumar et al. (NAR (2019) 47, 10059), \"... dsRNA expressed in neurons can silence a target gene in somatic tissues such as the intestine, muscle, and hypodermic (refs 8, 17, 18) and in the germline (19).\" Refs 8, 17, 18 are as follows:\n8. Jose A.M., Smith J.J., Hunter C.P. Export of RNA silencing from C. elegans tissues does not require the RNA channel SID-1. Proc. Natl. Acad. Sci. U.S.A. 2009; 106:2283–2288.\n17. Jose A.M., Garcia G.A., Hunter C.P. Two classes of silencing RNAs move between C. elegans tissues. Nat. Struct. Mol. Biol. 2011; 18:1184–1188.\n18. Raman P., Zaghab S.M., Traver E.C., Jose A.M. The double-stranded RNA binding protein RDE-4 can act cell autonomously during feeding RNAi in C. elegans. Nucleic Acids Res. 2017; 45:8463–8473.\n\nThe authors state that neurons do not express SID-1. This is not likely the case based on the analysis of neuron-specific gene expression (CeNGEN). Jose et al. have also shown that SID-1 is not required for the export of dsRNA to other tissues (though it is required for the import).\n\nMoreover, in their so-called \"intestine-specific\" RNAi experiments, the authors claim they are \"confirming\" their results by using a sid-1 mutant background, but in fact because of the possibility of spreading, their initial experiments are not meaningful. The only tissue-specific RNAi experiments performed by the authors that use generally accepted methodology are limited to those using the VP303 strain in the analysis of intestine-specific insulins, as noted in my initial review. It may be helpful for the authors to review the rationale for the use of these strains (and other strains utilized for tissue-specific RNAi). To be clear, the issue is not \"off-target effects\" stated by the authors in their response, but rather, the targeted gene will be affected in a tissue and cell type distinct from what was intended.\n\nThe authors refer to \"multiple orthogonal lines of evidence,\" but the only ones that relate to neuron-specific issues may be the use of cell-specific rescue, which is spotty and only done in limited instances, and in any case, does not establish necessity, only supports sufficiency. The key results derived from what the authors perceived were neuron-specific RNAi experiments are not supported by \"multiple orthogonal lines of evidence.\"\n\nThe authors refer to their prior 2017 study that utilized the same methodology. I am not aware of other manuscripts using the same method, though the concerns stated here would still apply. Given the experimental concerns outlined here, the authors' 2017 manuscript also invites considerable skepticism.", "reviewer_2": "The authors fully addressed my questions in their revised manuscript.", "reviewer_3": "I think that the authors have adequately addressed the comments made by the reviewers in the revised version of the manuscript. Therefore, I have no further comments.", "reviewer_4": "The authors have addressed my concerns for the most part. I remain a bit skeptical of the overall model for the reasons stated in my original review, but if the authors are convinced then perhaps it makes sense to publish and let the field digest the results.", "reviewer_5": "Reviewer #1 (Remarks to the Author):\nLei et al. present a study that examines the role of gut-to-brain signaling in the simple\nanimal C. elegans in modulating decision-making and behavioral responses to\nbacterial food cues and pathogenicity. The manuscript gets off to a strong start. The\nauthors set up a nice assay that assesses the “risk-taking” behavior in crossing a\nnoxious chemical barrier to access a food signal. The authors then examine how\nprior exposure to nutritious bacteria as well as three pathogenic bacteria, and also\nthe effect of bacterial food removal, influence this decision making. The authors carry\nout a number of solid control experiments and confirm that fasting has a marked\neffect on subsequent behavioral responses. The authors find that exposure to\npathogenic Pseudomonas aeruginosa reduces the animal’s risk-taking food-seeking\nbehavior. Genetic experiments with tph-1 suggest a role for TPH-1 and the ADF\nneurons in mediating this response. The authors then conduct calcium-imaging\nexperiments of ADF responses to show how ADF activity is influenced during\nconditions of fasting and Pseudomonas exposure. The authors use a candidate\nexpression approach to identify a putative receptor, SRI-36, that is strongly induced\nin the ADF neurons by fasting and even more so by Pseudomonas infection. The\nauthors show that ADF responses are depending on AAK-2, DAF-16, and the NSY-1-\nSEK-1 p38 pathway. Up until this point, the manuscript is quite impressive with a\ncombination of beautiful genetics and imaging.\n\nHowever, once the manuscript begins to address site-of-action issues, there are\nserious concerns with the underlying method being used. The authors claim to be\ncarrying out “cell-specific RNAi.” Methods are somewhat cursory, but it appears that\nthe primary method being utilized is to use cell- or tissue-specific promoters to\nexpress + and – strands of a gene of interest off of a plasmid that has been\nintroduced into cells of interest by the generation of transgenic strains. What is not\ntaken into account is that this method does not take into account the ability for\ndsRNA to spread from cell-to-cell. For this reason, cell-specific RNAi methods are\ngenerally performed in an RNAi-defective strain with cell-specific rescue of RNAi\n(e.g., rde-1). The VP303 strain used for the screen for intestine-specific insulins has\nthese attributes. But none of the other ostensibly cell-specific RNAi experiments\nutilize this strain or have any indication of a cell-specific rescue of RNAi activity.\nThus, one cannot conclude anything about cell necessity from these site-of-action\nexperiments. (It appears that the author used this method in a prior 2020 Nature\nCommunications article as well.)\nUnless there is a trivial explanation that I am missing, this basic experimental\noversight seriously undermines the confidence of this reviewer in the basic\nmethodology and rigor of the manuscript. This is especially disappointing as the first\npart of the manuscript is remarkably impressive.\n\nReviewer #2 (Remarks to the Author):\n“Intestinal pathogens override hunger-driven decision-making via immuneregulation\nof central serotonin signaling in C. elegans”\nSerotonin is known to react to various environmental stresses and internal states to internal states to\nmodulate neural functions and generate behavioral responses. Some of these\nbehavioral responses are orthogonal when the conditions are presented alone to the\nanimals. How serotonin signaling regulates neural activity to produce coherent and\nbalanced behavioral decisions under conditions demanding conflicting outputs from\nthe nervous system is a fundamentally important but challenging question. In the\nabove manuscript, Lei and Chen et al. set out to address this issue.\nThe authors employed a risk-taking decision paradigm in C. elegans where the\nworms’ attraction to an attractive odorant diacetyl (sensed by AWA sensory neuron)\nis hindered by a hyperosmotic glycerol barrier (sensed by ASH sensory neuron).\nThey characterized how serotonin signaling modulated this decision-making process\nunder two well-controlled conditions. While starvation promotes risk-taking as\npreviously shown, the authors found that feeding on a pathogenic bacteria\nPseudomonas aeruginosa PA14 suppressed the effect of starvation and inhibited the\ndecision to cross the hyperosmotic barrier.\nUsing this assay together with cell-specific manipulation of serotonin production and\nneural activity, the authors showed that starvation and P. aeruginosa infection\nconverged on the serotonin signal produced by ADF neurons to regulate behavior\ndecision on diacetyl/glycerol response, and that starvation and infection both induced\nADF response to diacetyl. Using previously published expression profiles, RNAi, and\ncalcium imaging, CRISPR and cell-specific rescue, the authors further showed that\nstarvation and P. aeruginosa infection upregulated the expression of a GPCR sri-36\nin ADF and that sri-36 mediated diacetyl responses in fasted and infected worm in\nADF neurons and in behavioral decision. The authors further identified two intestinal\npathways, the aak-2/daf-16 pathway and pmk-2/daf-16 pathway, that upregulated sri-\n36 under fasting and infection conditions. These pathways upregulated expression of\ntwo insulin-like peptides, ins-37 and ins-7, in fasted and infected worms to modulate\nADF expression of sri-36 and the behavioral decision between diacetyl/glycerol.\nFurthermore, the authors mapped the downstream neural circuit and serotonin\nreceptors that mediated starvation and infection-regulated diacetyl response in ADF\nand behavioral decision, and demonstrated that a stronger release of serotonin from\nADF in infected worms compared with starved worms suppressed the risk-taking\neffect of starvation. Based on these findings, they delineate a molecular strategy in a\nneuronal circuit that integrates food availability and infection to produce a balanced\nbehavior decision between safety-seeking or risk-taking. The authors fully leverage\nthe experimental power of C. elegans by taking multiple approaches to address the\nquestion at molecular, cellular and circuit levels. The findings are in general\ncompelling. I have a few suggestions that will help complete the analysis.\n1. Is sri-36 expressed in any neurons other than ADF? The images in Figures 3g and\n4d, 4f are too dark to see clearly. The authors should provide a more complete\npresentation on Psri-36::GFP expression.\n2. The GCaMP6 responses in ADF are relatively small in amplitude – do the\ntransgenic worms expressing GCaMP6 in ADF neuron display normal behavioral\ndecisions in the diacetyl/glycerol paradigm? Also importantly, the authors need to\nprovide their methods for calcium imaging, one of the major experimental\napproaches in this paper.\n3. The authors need to document their source of P. aeruginosa PA14 strain.\n4. In Figure 5d, it should be ins-7 rescue, not inx-7 rescue.\n\nReviewer #3 (Remarks to the Author):\nThe authors show that fasting and intestinal infection with Pseudomonas aeruginosa\nPA14 produce opposite decisions in a C. elegans multisensory conflict assay by\nacting through distinct gut–brain endocrine pathways that converge on the same\nsensory neurons. Fasting engages intestinal AAK-2/DAF-16 to induce INS-37,\nwhereas PA14 activates intestinal PMK-2 to induce INS-7; both act on ADF neurons\nvia DAF-2 to upregulate the chemoreceptor SRI-36, but bias serotonin output toward\napproach or avoidance. The authors have an present impressive amount of work that\ndefines a novel gut-brain communication pathway that dynamic sensory receptor\ntuning and divergent behavioral outcomes, offering a clear framework for how\ninternal state reshapes sensory processing and decision-making. The depth of\nanalysis is impressive including, identification chemoreceptor, Ca2+ imaging data\nand network analysis. The manuscript is clearly written and presents a compelling\nand conceptually novel gut–brain–behavior axis. I only have minor comments that\ncould be clarified.\n1. Other pathogens (S. aureus and EPEC) have no effect (Fig. 1b):\nThe mechanistic basis for this difference is unclear. Do these strains fail to colonize\nor persist after fasting, or do they not activate PMK-2 signaling?\n2. Colonization vs pathogenicity:\nCould it be that persistent gut bacteria could serve as internal “food-present” cues\nthat suppress hunger responses? It would be informative to test a nonpathogenic\nstrain that persists after fasting but does not activate PMK-2, to see if it fails to\nsuppress escape.\n3. PA14 under well-fed conditions (Supplementary Fig. 1b):\nThe authors report that PA14 does not alter decision-making in well-fed animals, but\nit is possible that PA14 still induces PMK-2 immune signaling under these conditions.\nIf so, the model might predict some reduction in risk-taking under a broader range of\nassay conditions, such as lower glycerol or higher diacetyl concentrations?\n4. 4 h vs 24 h infection (Supplementary Fig. 1g) and peptone supplementation (Fig.\n1d):\nSimilar escape suppression seen after 4 h and 24 h PA14 exposure (S1g) is\nintriguing, as longer exposure might be expected to increase colonization and PMK-2\nactivation.\n5. Residual fasting effect without 5-HT or ADF:\nEven with tph-1 loss (Fig. 1h), ADF silencing (Fig. 1j), or sri-36 deletion (Fig. 3f),\nfasting-induced escape remains higher than in well-fed animals.\n6. mod-5(n822) mutant phenotype:\nmod-5(n822) mutants, which have elevated extracellular 5-HT, show exaggerated\nfasting-induced escape (Fig. 1i). This seems inconsistent with the model, which\npredicts that high 5-HT should suppress escape in fasted worms.\n7. Exogenous 5-HT tests:\nIt could be informative to test exogenous 5-HT supplementation in WT and tph-1\nmutants under well-fed conditions, to see if low doses mimic fasting (increased\nescape) and high doses mimic PA14 infection (suppressed escape).\n8. sri-36 expression levels:\nThe “moderate vs high” SRI-36 expression model could be strengthened by\nexpressing sri-36 in ADF at moderate and high levels in sri-36 mutants.\n9. ILP release mutants:\nhid-1 and egl-3 loss abolish ADF responses (Fig. 5a), consistent with the model that\nintestinal ILPs act via ADF DAF-2, unc-31(e169) mutants show no effect on ADF\nresponses in any state (Fig. 2c). Does this mean that unc-31 is not requires for INS-\n37/7 release form the intestine?\n10. Other minor: Supplementary Fig. 2 is not referenced in the main text.\n11. line 295 The authors shoud weaken the statement : “These results demonstrate\nthat hunger and P. aeruginosa infection induce intestinal INS-37 and INS-7 release,\nrespectively” to indicate are consistent with.\n12. Different bacterial strains were cultured on distinct media and under different\nincubation conditions.\n13. For exogenous 5-HT stimulation, the authors applied serotonin directly to exposed\nneuronal somas via pressure ejection after incision. This is technically challenging.\n14. For Fig. S1f, fasting typically increases locomotion speed and roaming, which is\nnot observed here?\n15. Supplementary Fig. 2 is not referenced in the main text.\n\nReviewer #4 (Remarks to the Author):\nIn this submitted manuscript, Lei, Chen et al examine how C. elegans behavioral\ndecisions are modulated by internal state. They use a classic paradigm where worms\nneed to cross an aversive barrier to get to a chemoattractant Diacetyl. The authors\nfirst show that fasted animals cross the barrier more frequently, whereas P.\naeruginosa-infected animals that are fasted do not. They find that these behavioral\nchanges are dependent on serotonin production by ADF neurons. ADF is shown to\ndirectly respond to diacetyl, scaling up its response in fasted animals and even\nfurther upon infection. The authors then identify the sri-36 chemoreceptor as an ADF\nchemoreceptor that is upregulated upon fasting and even further upon infection. They\nshow that the fasting effects work through AAK-2 and Insulin signaling, whereas the\ninfection effects work through PMK/NSY/SEK signaling. Finally, the authors show\nthat the fasting effects require the interneurons AIB/AUA/URX, whereas the infection\neffects require AIA/AIY. Serotonin receptors in these interneurons are identified.\nMost of the analyses in this paper are well done. I appreciated the identification of\nSRI-36 as an induced chemoreceptor and the important role of ADF in this behavior.\nHowever, I have two main reservations about publication of this study in Nature\nCommunications:\n1) I worry that the final conceptual model in the paper doesn’t quite make sense in\nthe context of the field. First, the authors argue for a biphasic curve where ADF\npromotes escape when it releases a medium amount of serotonin, but not when it is\ninactive or releasing a lot of serotonin. It’s just not clear how this could work.\n2) Regarding the first half of the paper – I find these results convincing and I\nappreciate the work that the authors put into this. But this is very well traveled\nground. The idea that chemoreceptors are induced by fasting and infection has been\nestablished.\nI also have a few other minor comments:\n1. For the sri-36 gene expression studies, it would be preferable to use a CRISPR\nknock-in reporter.\n2. Is pmk/sek signaling involved in the fasting effects? And is aak-2 involved in the\ninfection effects?\n3. ADF releases other neurotransmitters – are they involved in these behaviors?\n\n[Second round of review]\nReviewer #1 (Remarks to the Author):\nThe revised manuscript by Liu and colleagues does not address the principal\nconcern raised in my initial review. The authors do not seem to grasp the seriousness\nof their lack of understanding of their own experimental system.\nThe authors state that their method is one that is widely accepted. In fact, the issue\nof whether dsRNA can spread from neurons has been examined by Jose and Hunter\nand colleagues. Specifically, as noted by Ravikumar et al. (NAR (2019) 47, 10059),\n\"... dsRNA expressed in neurons can silence a target gene in somatic tissues such\nas the intestine, muscle, and hypodermic (refs 8, 17, 18) and in the germline (19).\"\nRefs 8, 17, 18 are as follows:\n8. Jose A.M., Smith J.J., Hunter C.P. Export of RNA silencing from C. elegans tissues\ndoes not require the RNA channel SID-1. Proc. Natl. Acad. Sci. U.S.A. 2009;\n106:2283–2288.\n17. Jose A.M., Garcia G.A., Hunter C.P. Two classes of silencing RNAs move\nbetween C. elegans tissues. Nat. Struct. Mol. Biol. 2011; 18:1184–1188.\n18. Raman P., Zaghab S.M., Traver E.C., Jose A.M. The double-stranded RNA\nbinding protein RDE-4 can act cell autonomously during feeding RNAi in C. elegans.\nNucleic Acids Res. 2017; 45:8463–8473.\nThe authors state that neurons do not express SID-1. This is not likely the case\nbased on the analysis of neuron-specific gene expression (CeNGEN). Jose et al.\nhave also shown that SID-1 is not required for the export of dsRNA to other tissues\n(though it is required for the import).\nMoreover, in their so-called \"intestine-specific\" RNAi experiments, the authors claim\nthey are \"confirming\" their results by using a sid-1 mutant background, but in fact\nbecause of the possibility of spreading, their initial experiments are not meaningful.\nThe only tissue-specific RNAi experiments performed by the authors that use\ngenerally accepted methodology are limited to those using the VP303 strain in the\nanalysis of intestine-specific insulins, as noted in my initial review. It may be helpful\nfor the authors to review the rationale for the use of these strains (and other strains\nutilized for tissue-specific RNAi). To be clear, the issue is not \"off-target effects\"\nstated by the authors in their response, but rather, the targeted gene will be affected\nin a tissue and cell type distinct from what was intended.\nThe authors refer to \"multiple orthogonal lines of evidence,\" but the only ones that\nrelate to neuron-specific issues may be the use of cell-specific rescue, which is\nspotty and only done in limited instances, and in any case, does not establish\nnecessity, only supports sufficiency. The key results derived from what the authors\nperceived were neuron-specific RNAi experiments are not supported by \"multiple\northogonal lines of evidence.\"\nThe authors refer to their prior 2017 study that utilized the same methodology. I am\nnot aware of other manuscripts using the same method, though the concerns stated\nhere would still apply. Given the experimental concerns outlined here, the authors'\n2017 manuscript also invites considerable skepticism.\n\nReviewer #2 (Remarks to the Author):\nThe authors fully addressed my questions in their revised manuscript.\n\nReviewer #3 (Remarks to the Author):\nI think that the authors have adequately addressed the comments made by the\nreviewers in the revised version of the manuscript. Therefore, I have no further\ncomments.\n\nReviewer #4 (Remarks to the Author):\nThe authors have addressed my concerns for the most part. I remain a bit skeptical\nof the overall model for the reasons stated in my original review, but if the authors are\nconvinced then perhaps it makes sense to publish and let the field digest the results."}, "author_response": "Response to Reviewers’ Comments\nWe thank the reviewers for their careful and constructive comments, which have\nsubstantially improved our manuscript. We are pleased that the revisions have\naddressed the concerns raised. Below, we provide point-by-point responses. For\nclarity, reviewer comments are reproduced in italics, followed by our responses, with\nreference to the relevant text in the revised manuscript. All changes have been\nincorporated into the revised version and are highlighted in blue.\n\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe revised manuscript by Liu and colleagues does not address the principal\nconcern raised in my initial review. The authors do not seem to grasp the seriousness\nof their lack of understanding of their own experimental system.\n\nThe authors state that their method is one that is widely accepted. In fact, the issue\nof whether dsRNA can spread from neurons has been examined by Jose and Hunter\nand colleagues. Specifically, as noted by Ravikumar et al. (NAR (2019) 47, 10059),\n\"... dsRNA expressed in neurons can silence a target gene in somatic tissues such\nas the intestine, muscle, and hypodermic (refs 8, 17, 18) and in the germline (19).\"\nRefs 8, 17, 18 are as follows:\n8. Jose A.M., Smith J.J., Hunter C.P. Export of RNA silencing from C. elegans tissues\ndoes not require the RNA channel SID-1. Proc. Natl. Acad. Sci. U.S.A. 2009;\n106:2283–2288.\n17. Jose A.M., Garcia G.A., Hunter C.P. Two classes of silencing RNAs move\nbetween C. elegans tissues. Nat. Struct. Mol. Biol. 2011; 18:1184–1188.\n18. Raman P., Zaghab S.M., Traver E.C., Jose A.M. The double-stranded RNA\nbinding protein RDE-4 can act cell autonomously during feeding RNAi in C. elegans.\nNucleic Acids Res. 2017; 45:8463–8473.\n\nThe authors state that neurons do not express SID-1. This is not likely the case\nbased on the analysis of neuron-specific gene expression (CeNGEN). Jose et al.\nhave also shown that SID-1 is not required for the export of dsRNA to other tissues\n(though it is required for the import).\n\nMoreover, in their so-called \"intestine-specific\" RNAi experiments, the authors claim\nthey are \"confirming\" their results by using a sid-1 mutant background, but in fact\nbecause of the possibility of spreading, their initial experiments are not meaningful.\nThe only tissue-specific RNAi experiments performed by the authors that use\ngenerally accepted methodology are limited to those using the VP303 strain in the\nanalysis of intestine-specific insulins, as noted in my initial review. It may be helpful\nfor the authors to review the rationale for the use of these strains (and other strains\nutilized for tissue-specific RNAi). To be clear, the issue is not \"off-target effects\"\nstated by the authors in their response, but rather, the targeted gene will be affected\nin a tissue and cell type distinct from what was intended.\n\nThe authors refer to \"multiple orthogonal lines of evidence,\" but the only ones that\nrelate to neuron-specific issues may be the use of cell-specific rescue, which is\nspotty and only done in limited instances, and in any case, does not establish\nnecessity, only supports sufficiency. The key results derived from what the authors\nperceived were neuron-specific RNAi experiments are not supported by \"multiple\northogonal lines of evidence.\"\n\nThe authors refer to their prior 2017 study that utilized the same methodology. I am\nnot aware of other manuscripts using the same method, though the concerns stated\nhere would still apply. Given the experimental concerns outlined here, the authors'\n2017 manuscript also invites considerable skepticism.\n\nResponse:\nWe thank the reviewer for continued engagement and for highlighting concerns\nregarding the interpretation of RNAi-based experiments. We agree that inter-tissue\nmovement of dsRNA has been documented, including the work from Ravikumar et al.\nWe appreciate the opportunity to clarify both the scope of our conclusions and the\nrole of RNAi in this study. First, our primary conclusions regarding intestinal INS-37\nand INS-7 signaling are based on experiments using the intestine-specific RNAi\nstrain VP303, which is widely accepted for restricting RNAi efficacy to the intestine.\nThese experiments form the foundation of our claims regarding intestinal ILP\nregulation. Second, conclusions related to neuronal mechanisms are not based on\nRNAi alone. Rather, RNAi-based perturbations are interpreted in conjunction with\nmultiple independent approaches, including loss-of-function mutants, tissue-specific\nrescue experiments, CRISPR knock-in reporters, and direct functional measurements\nof neuronal activity and behavior. We agree that cell-specific rescue experiments\nestablish sufficiency rather than necessity, and we have therefore framed these\nresults as supportive rather than definitive on their own.\n\nIn response to the reviewer’s concerns and following editorial guidance, we have\nnow explicitly discussed the limitations of cell-restricted RNAi approaches and the\npotential contribution of low-level silencing in unintended tissues in the revised\nDiscussion as follows: “A limitation of this study is the use of RNAi to infer cell-\nspecific gene function. Previous studies have shown that dsRNA can spread between\ntissues65, and therefore low-level silencing in unintended cells cannot be excluded.\nAccordingly, RNAi-based results were interpreted in conjunction with complementary\napproaches, including genetic mutants, tissue-specific rescue, CRISPR knock-in\nreporters, and functional imaging and behavioral analyses.”\n\nWe hope that this clarification, together with the added Discussion text, addresses\nthe reviewer’s concerns while accurately reflecting both the strengths and limitations\nof the experimental approaches used in this study.\n\nReviewer #2 (Remarks to the Author):\nThe authors fully addressed my questions in their revised manuscript.\nResponse: We are pleased that the revisions have fully addressed the reviewer’s\nquestions.\n\nReviewer #3 (Remarks to the Author):\nI think that the authors have adequately addressed the comments made by the\nreviewers in the revised version of the manuscript. Therefore, I have no further\ncomments.\nResponse: We thank the reviewer for this positive assessment and are pleased that\nthe revisions adequately address the comments raised.\n\nReviewer #4 (Remarks to the Author):\nThe authors have addressed my concerns for the most part. I remain a bit skeptical\nof the overall model for the reasons stated in my original review, but if the authors are\nconvinced then perhaps it makes sense to publish and let the field digest the results.\nResponse: We appreciate the reviewer’s careful evaluation of the revised\nmanuscript and their candid perspective. While we acknowledge that aspects of the\nproposed model will benefit from further investigation, we believe that the data\npresented provide a coherent and well-supported framework. We are grateful for the\nreviewer’s thoughtful consideration and are pleased that the revisions have\naddressed the major concerns raised."}]

Decision letter