Intercellular sphingolipid signaling mediates aversive learning in C. elegans

Yu-Chun Wu; Isabel Beets; Bennett William Fox; Diana Fajardo Palomino; Li Chen; Chien-Po Liao; Elke Vandewyer; Liang-Yi Lin; Chun-Wei He; Li-Tzu Chen; Chih-Ta Lin; Frank C Schroeder; Chun-Liang Pan

doi:10.1016/j.cub.2025.03.082

. Author manuscript; available in PMC: 2026 Apr 18.

Published in final edited form as: Curr Biol. 2025 Apr 18;35(10):2323–2336.e9. doi: 10.1016/j.cub.2025.03.082

Intercellular sphingolipid signaling mediates aversive learning in C. elegans

Yu-Chun Wu ^1,², Isabel Beets ^3,⁵, Bennett William Fox ^4,⁵, Diana Fajardo Palomino ⁴, Li Chen ³, Chien-Po Liao ^1,^2,⁶, Elke Vandewyer ³, Liang-Yi Lin ¹, Chun-Wei He ^1,^2,⁷, Li-Tzu Chen ^1,^2,⁸, Chih-Ta Lin ¹, Frank C Schroeder ⁴, Chun-Liang Pan ^1,^2,^9,^*

PMCID: PMC12799178 NIHMSID: NIHMS2130171 PMID: 40252647

SUMMARY

Physiological stress in non-neural tissues drives aversive learning for sensory cues associated with stress. However, the identities of signals derived from non-neural tissues and the mechanisms by which these signals mediate aversive learning remain elusive. Here, we show that intercellular sphingolipid signaling contributes to aversive learning under mitochondrial stress in C. elegans. We found that stress-induced aversive learning requires sphingosine kinase, SPHK-1, the enzyme that produces sphingosine-1-phosphate (S1P). Genetic and biochemical studies revealed an intercellular signaling pathway in which intestinal or hypodermal SPHK-1 signals through the neuronal G protein-coupled receptor, SPHR-1, and modulates responses of the octopaminergic RIC neuron to promote aversive learning. We further show that SPHK-1-mediated sphingolipid signaling is required for learned aversion of Chryseobacterium indologenes, a bacterial pathogen found in the natural habitats of C. elegans, which causes mitochondrial stress. Taken together, our work reveals a sphingolipid signaling pathway that communicates from intestinal or hypodermal tissues to neurons to promote aversive learning in response to mitochondrial stress and pathogen infection.

In brief

Wu et al. identify a sphingolipid signaling pathway that mediates aversive learning under mitochondrial stress in C. elegans. Sphingolipids target the octopaminergic neurons, altering their response properties to drive avoidance behavior. This pathway is important for learned aversion of Chryseobacterium indologenes, a natural C. elegans pathogen.

Graphical Abstract

graphic file with name nihms-2130171-f0001.jpg

INTRODUCTION

Changes in internal states, such as hunger or sex drive, motivate animal behaviors to restore homeostasis.^1,2 Tissue damage can also drive aversive learning and avoidance to minimize exposure to pathogens and environmental hazards.^3–6 Supporting this notion, in C. elegans and Drosophila, pathogen virulence and toxicity are essential for learned avoidance of pathogen-associated sensory cues.^3,7–9 This implies that non-neural tissue damage alters nervous system functions via intercellular signals.¹⁰ While a subset of metabolites and hormones that signal the nutritional states for behavioral modulation has been identified,^10–12 those that report physiological disruption from non-neural tissues to the brain in aversive learning remain incompletely defined.

As essential organelles, mitochondria are a prime target for microbial attack.^13,14 Therefore, the integrity of mitochondria is closely monitored by the host to detect potential pathogen invasion.¹⁴ In C. elegans, mitochondrial disruption or infection by the pathogen Pseudomonas aeruginosa (P. aeruginosa) shares activation of xenobiotic and immunity genes, supporting the utility of mitochondrial disruption as a proxy for pathogen infection.¹⁵ Interestingly, ceramide and sphingolipid biosynthesis is exploited by the host cell to signal mitochondrial injury and activate stress responses.^14,16 In addition to intracellular signaling, sphingosine-1-phosphate (S1P) also mediates intercellular signaling by binding its cell surface G protein-coupled receptors (GPCRs), the S1P receptors (S1PR1–5).^17,18 S1PRs are expressed in most mammalian tissues, including the brain, suggesting S1P could be an inter-tissue signal for physiological homeostasis.¹⁹ S1PR signaling has been implicated in various neurological disorders.¹⁹ For example, microglial-derived S1P increases in the infarcted area of murine model of brain ischemia.²⁰ All five S1PRs are expressed in microglia, and signaling through S1PRs displays complex regulatory roles in neuroimmune disorders.²¹ A study in Drosophila shows that the glial cells convert excess very long-chain fatty acids to S1P; secreted S1P then recruits immune cells and causes neurotoxic effects.²² These examples illustrate the importance of intercellular sphingolipid signaling in the brain, but whether it also regulates behavioral adaptation to systemic mitochondrial stress remains unexplored.

We recently established a C. elegans paradigm of stress-induced aversive learning, based on the pioneering study of Melo and Ruvkun.^5,23–26 In this model, aversive learning is triggered by disruption of mitochondrial functions in the intestine and hypodermis.^5,23–25 Here, we report that intercellular sphingolipid signaling communicates from the intestine and hypodermis to the nervous system, relaying information of compromised mitochondrial integrity to a central modulatory neuron to promote aversive learning. Importantly, this pathway is required for learned aversion of Chryseobacterium indologenes (C. indologenes), a natural pathogen of C. elegans. Our work is an entry point to dissect the mechanisms of gut/hypodermal-brain sphingolipid signaling in state-dependent learning and behaviors.

RESULTS

SPHK-1/sphingosine kinase regulates stress-induced aversive learning

Mitochondrial stress, such as that induced by antimycin A, a cytochrome c reductase inhibitor, triggers C. elegans to exhibit avoidance behavior and leave the food bacteria that are otherwise attractive (Figures 1A and 1B).⁵ We recently showed that this stress-induced bacterial avoidance is a form of aversive learning.^23–26 In C. elegans, sphingolipid pathways monitor the integrity of mitochondrial functions.¹⁴ Avoidance of food bacteria OP50 E. coli under mitochondrial stress is reduced by blocking sphingolipid biosynthesis,¹⁴ suggesting that sphingolipid signaling promotes avoidance behavior under stress. We first screened mutants putatively required for the synthesis of sphingolipids (Figure 1C), including serine C-palmitoyltransferases (sptl-1, sptl-2), sphingosine N-acyltransferases (hyl-1, hyl-2, lagr-1), and sphingosine kinase (sphk-1).²⁷ Stress-induced bacterial avoidance was reduced in the sptl-, sptl-2, hyl-2, and sphk-1 mutants (Figures 1B and S1A). In contrast, hyl-1 and lagr-1 did not seem to play significant roles (Figure S1A).

Figure 1. — (A) Bacterial avoidance assay. Animals are placed on a bacterial lawn with vehicle or antimycin A, and the avoidance index is quantified after 6 h.

(B) Bacterial avoidance of wild-type and the *sphk-1* mutants.

(C) Sphingolipid biogenesis. Palmitoyl-coenzyme A (CoA) and isoC15-CoA are used by vertebrates and nematodes, respectively.

(D) Gene and protein structures of *sphk-1a*.

(E and F) High-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS). Structures (left) and quantification (right) of sphingosine (E) and sphinganine (F) are shown. *C. elegans* sphingosine and sphinganine are 17C and iso-branched, unlike the 18C sphingolipids of vertebrates. The chemical formulas represent protonated forms of sphingolipids. n = 2 for *sphk-1*/antimycin and 3 for the other genotype/condition combinations. Bars are mean ± standard error of mean (SEM). Unpaired t test.

(G) Bacterial avoidance of the *sphk-1* mutant with exogenous S1P or ceramide.

For (B) and (G), the results of independent experiments are shown, with median ± quartiles and minimum/maximum whiskers. n = numbers of independent experiments, with 50–200 animals per experiment. Two-way ANOVA followed by Bonferroni’s correction. These parameters also apply to similar experiments, including bacterial chemotaxis, in Figures 2, 3, 4, 5, and 6.

See also Figure S1.

We focused on sphk-1 as it represents a bottleneck of ceramide biosynthesis. sphk-1 encodes a sphingosine kinase that converts sphingosine to S1P (Figure 1C), and is shown to regulate mitochondrial stress responses in C. elegans.¹⁶ The sphk-1(ok1097) deletion lacks the kinase and calmodulin-binding domains of SPHK-1 (Figure 1D). sphk-1 mutants exhibited decreased avoidance (Figure 1B) but also moved more slowly (Figure S1B), although other locomotion parameters were unaffected (Figures S1C and S1D). In the metabolomes resolved by high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS), sphingosine and sphinganine, which are substrates of SPHK-1, accumulated in the sphk-1 mutant, corroborating the role of SPHK-1 in sphingosine metabolism (Figures 1E and 1F). Other major lipid derivatives, such as lysolipids and ascarosides, were not significantly different in the sphk-1 mutant, suggesting a specific defect in sphingosine biosynthesis (Figures S1E–S1G). Furthermore, exogenous S1P fully rescued the behavioral defects of the sphk-1 mutant, whereas ceramide failed to rescue these defects (Figure 1G). Therefore, sphingosine metabolism via SPHK-1 is important for aversive learning under mitochondrial stress.

S1P acts in aversive learning under stress

To specifically examine aversive learning, we used the bacterial chemotaxis assay (Figure 2A). Low levels of antimycin converted innate preference of C. elegans for OP50 to aversion (Figure 2B). We showed previously that this acquired bacterial aversion is associated with mitochondrial stress in non-neural tissues and persists for up to 15 h after antimycin withdrawal (Figures 2A–2C and S1H),²³ suggesting that mitochondrial stress induces long-term memory. Of note, the aversive behavior was gradually lost if OP50-trained animals were kept on the same OP50 bacteria after stress removal (Figure S1H), which suggests extinction, a characteristic of associative memory. The sphk-1 mutant was defective in stress-induced aversion of OP50 (Figures 2B and 2C). Also, 3 h of S1P treatment before antimycin training, but not before chemotaxis testing, fully rescued the chemotaxis phenotype of the sphk-1 mutant (Figure 2C). These data indicate that S1P is important for aversive learning. Moreover, in wild-type animals, both S1P and ceramide enhanced bacterial avoidance under mild mitochondrial stress but did not trigger aversive behavior without stress, suggesting that their effects on behavior are stress specific (Figure 2D). The sphk-1 mutant displayed normal chemotaxis to bacterial cues or the non-associative odor benzaldehyde (Figures 2B and S1I) without stress. Finally, using a lower concentration of antimycin (1.125 μM) that induces modest avoidance and therefore serves as a sensitized condition for enhancing aversive behavior,^23,25 we showed that genetic disruption of spl-1 and spl-2, S1P lyases that degrade S1P (Figure 1C),²⁸ enhanced stress-induced avoidance (Figure S1J). Taken together, these results imply that S1P signaling instructs aversive learning under stress.

Mitochondrial localization of SPHK-1 is required for aversive learning

To visualize endogenous SPHK-1, we inserted a linker and GFP into the endogenous sphk-1 locus using CRISPR-Cas9 editing (Figure 2E).²⁹ We found that sphk-1 was expressed in the intestine, hypodermis, and neurons, consistent with a recent study (Figure S1K).¹⁶ The subcellular localization of sphingosine kinase is important for S1P signaling,^28,30 and SPHK-1::GFP expressed from a multi-copy gene array is recruited to the mitochondria under paraquat-induced oxidative stress.¹⁶ Endogenous SPHK-1 was diffused in the cytosol of the intestinal cells but was redistributed to the mitochondrial outer membrane under antimycin treatment (Figures 2F and 2G), consistent with prior studies.¹⁶ Complementation of the sphk-1 mutant in the intestine or hypodermis, but not neurons, fully restored stress-induced avoidance (Figure 2H), suggesting that sphk-1 functions in non-neural tissues to regulate aversive learning. Rescue requires the kinase domain and the calmodulin-binding domain that targets SPHK-1 to the mitochondria (Figure 2I), suggesting that both the enzymatic activity and mitochondrial localization of SPHK-1 are important.

Lipocalin LPR-3 is an S1P chaperone in aversive learning

We next investigated potential roles of S1P chaperones in stress-induced aversive learning.³¹ Lipocalins are lipid chaperones for cholesterol, phospholipids, and sphingolipids.³² The S1P chaperone apolipoprotein M (ApoM) is structurally similar to mammalian lipocalin 2,^19,33,34 suggesting that lipocalins facilitate the extracellular transport of S1P. We silenced all seven C. elegans lipocalin genes and found that RNAi of lpr-1 or lpr-3 reduced antimycin-induced avoidance (Figure 3A). As lpr-1 mutants display penetrant larval lethality,³⁵ we focused on lpr-3. As reported previously, lpr-3 was highly expressed in larvae but undetectable in adult (Figure S2A).³⁶ lpr-3 upregulation under stress was confirmed by RT-qPCR (Figure 3B). Using a functional ssSfGFP::LPR-3 transgene,³⁶ we verified the increase of LPR-3 protein by antimycin, using fluorescent microscopy and western blotting (Figures 3C–3E and S3A–S3E). These results confirmed an important role for LPR-3 in stress-induced aversive learning.

Figure 3. — (A) Bacterial avoidance under RNAi of the lipocalin (*lpr*) genes. Two-way ANOVA followed by Bonferroni’s correction.

(B) *lpr-3* expression in young adults under antimycin by RT-qPCR. n = 3 experiments. Bars are mean ± SEM.

(C) Epifluorescent images of GFP::LPR-3 in young adults. Scale bar, 100 μm.

(D) Quantification of GFP intensity in (C). N = numbers of animals quantified from four independent cohorts.

(E) Normalized LPR-3 levels in young adults using western blot. n = 4 experiments. Bars are mean ± SEM. For (B), (D), and (E), unpaired t test.

(F) Bacterial avoidance assays. Two-way ANOVA followed by Bonferroni’s correction.

(G) LPR-3-S1P binding assay. Lysates of worms expressing GFP::LPR-3 were precipitated by GFP nanobodies. The precipitated GFP::LPR-3 was incubated with biotin-conjugated S1P. S1P-biotin bound to GFP::LPR-3 was detected by chemiluminescence using streptavidin-horseradish peroxidase (HRP) conjugates and a plate reader. Unlabeled S1P was added to compete with S1P-biotin for GFP::LPR-3 binding.

(H) Luminescence counts of S1P bound with LPR-3, with western blot for GFP and actin to calculate the GFP/actin ratio, against which read counts of S1P-biotin were normalized. n = 3 or 6 experiments. Bars are mean ± SEM. Ratio paired t test. Lysates from the strain CLP1306: *twnEx766(Pelt-2::GFP)*, which expresses free GFP in the intestine, were used as controls.

(I) Modeling of LPR-3 structure by AlphaFold2. The regions predicted to interact with S1P are indicated. Upper: structural alignment of human ApoM and *C. elegans* LPR-3. Lower: possible S1P-interacting residues of LPR-3. Pink, hydrophilic residues; green, hydrophobic residues. Blue, the alkyl chain of S1P. Atoms of the S1P head group are in red, gray, and orange.

See also Figures S2 and S3.

As many lpr-3 null mutants arrested as larvae,³⁶ we generated mosaic mutants with lpr-3 deletion in the intestine or hypodermis by somatic CRISPR-Cas9 (Figure S2B).³⁷ The knockout efficiency was confirmed by single-molecule fluorescence in situ hybridization (smFISH) (Figures S2C–S2E). Tissue-specific lpr-3 knockout did not impair locomotion (Figures S2F–S2H). We found that intestinal or hypodermal lpr-3 knockout reduced bacterial avoidance, suggesting that lpr-3 in non-neural tissues is required for aversive learning (Figure 3F). Tissue-specific lpr-3 knockout did not further decrease bacterial avoidance of the sphk-1 mutant, indicating that lpr-3 and S1P signaling function in a common pathway (Figure 3F). RNAi of lpr-1 did not further decrease the avoidance behavior of the hypodermal lpr-3 knockout animals (Figure S2I), suggesting that LPR-1 and LPR-3 act in the same pathway, possibly via forming heteromeric protein complexes.

To test whether LPR-3 is an S1P chaperone, we incubated lysates of the ssSfGFP::LPR-3 worms with biotin-conjugated S1P, followed by GFP nanobody-mediated LPR-3 precipitation and chemiluminescent detection by streptavidin-horseradish peroxidase (HRP) (Figure 3G). A worm strain expressing GFP served as the control (Figures 3H and S3F–S3H). The results showed that LPR-3 bound biotin-conjugated S1P (Figures 3H and S3F–S3H). Importantly, binding of S1P-biotin to LPR-3 was outcompeted by excess unlabeled S1P (Figures 3H and S3F–S3H), confirming the specificity of S1P-biotin and LPR-3 interaction. The AlphaFold2 model of LPR-3 implicates amino acids and structural elements in potentially binding S1P, based on its structural similarity to human ApoM (Figure 3I). We conclude that LPR-3 is a carrier for S1P under stress.

The C. elegans C24B5.1/sphr-1 encodes an S1PR

The human genome encodes five GPCRs of S1PRs that are broadly expressed and that regulate various cellular processes such as angiogenesis and immunity.¹⁹ The S1PRs in C. elegans remain unknown. Sequence analysis of several orphan C. elegans GPCRs and human S1PRs identified conserved amino acids for lipid binding and S1P engagement (Figure S4A).³⁸ Deletion mutants of three C. elegans GPCR genes (srsx-22, F10D7.1, and F57A8.4) displayed intact avoidance (Figures S4B and S4C). However, a CRISPR-Cas9-engineered frameshift deletion of sphr-1 (for S1PR-1, previously C24B5.1) decreased stress-induced aversive learning (Figures 4A–4C). The sphr-1 mutant displayed reduced speed and increased reversal (Figures S4D–S4F), yet it showed intact chemotaxis to non-associative odors or bacterial cues at baseline (Figures 4C and S4G–S4I), suggesting that the locomotion defects do not hamper gross sensory behaviors in the sphr-1 mutant. mCherry expression from the 6-kb sphr-1 promoter was detected in neurons, including RIC (marked by the tbh-1/tyramine β-hydroxylase promoter), a subset of the cholinergic motor neurons (marked by the unc-17/vesicular acetylcholine transporter promoter) and others in the head (Figure 4D). We recently showed that RIC promotes aversive learning under mitochondrial stress.²⁵ Learning deficits of the sphr-1 mutant were fully rescued by sphr-1 cDNA expressed from the endogenous or RIC promoter, but not in the cholinergic neurons (Figure 4E). These data suggest that sphr-1 functions in RIC to regulate aversive learning.

We confirmed that SPHR-1 is an S1PR with the following experiments. First, avoidance defects of the sphk-1; sphr-1 double mutant were comparable to those of the sphk-1 single mutant, suggesting that sphr-1 functions in the S1P pathway (Figure 4F). Next, we utilized a luminescence-based calcium mobilization assay to measure SPHR-1 activation in cultured cells (Figure S5A).^39,40 Gα₁₆, a promiscuous Gq, was used as an effector that couples GPCRs to phospholipase Cβ (PLCβ),^40–42 allowing us to measure GPCR activation using a calcium-based luminescence readout (Figure S5A). Chinese hamster ovary (CHO) cells transfected with Gα₁₆ and sphr-1 responded more robustly to S1P, compared with control Gα₁₆ cells without sphr-1 (Figure S5B). However, these control cells also displayed calcium responses, which could come from activation of endogenous S1PRs in this cell line (Figure S5B).⁴³ Supporting this idea, these background signals disappeared without Gα₁₆ transfection (Figure S5B). SPHR-1::GFP was indeed enriched at the plasma membrane of CHO cells (Figure S5C). Furthermore, two-electrode voltage-clamp (TEVC) recording showed that Xenopus oocytes expressing SPHR-1 displayed significantly greater membrane activation by S1P, compared with oocytes without SPHR-1 (Figure S5D). Finally, we showed that human S1PR2 or S1PR3 expression in RIC fully rescued the sphr-1 mutant for stress-induced aversive behaviors (Figures 4G and 4H).

The AlphaFold2 structure of SPHR-1 maps amino acids and grooves potentially interacting with S1P (Figure 4I).³⁸ To explore this further, the four amino acids of SPHR-1 lining the binding pocket for S1P alkyl chain were mutated to alanine. When expressed in RIC, this SPHR-1(I176A, V177A, T335A, L339A, or 4A) failed to rescue the avoidance defects of the sphr-1 mutant (Figure 4J). Taken together, these data strongly imply that SPHR-1 is a C. elegans S1PR.

Our results suggest that S1P targets RIC neurons to promote aversive learning. Indeed, genetic RIC ablation reduced avoidance behavior in the wild type as reported,²⁵ and it did not further worsen the behavioral deficits of the sphk-1 and only slightly affected the response of the sphr-1 mutant (Figure 4K). We previously found that octopamine from the RIC neurons regulates stress-induced aversive learning through the SER-6 GPCR.²⁵ The ser-6; sphr-1 double mutant showed reduced avoidance comparable with that of the ser-6 or sphr-1 single mutants (Figure S4J), further confirming that S1P acts through the RIC neuron, octopamine, and the SER-6 receptor.

Sphingolipid signaling remodels RIC properties under mitochondrial stress

We recently showed that RIC develops novel responses to bacterial cues under mitochondrial insults, which are otherwise absent in non-stressed animals.²⁵ To test whether S1P modulates RIC functions under stress, we performed calcium imaging in a microfluidic chamber.⁴⁴ OP50 supernatant evoked robust calcium responses in the distal RIC axon under stress, which were significantly reduced in the sphk-1 and sphr-1 mutants (Figures 5A, 5B, and S6A–S6C). Supplement of S1P restored evoked RIC activity in the sphk-1 mutant, indicating that sphk-1 modulates RIC responses through S1P (Figures 5C, 5D, and S6D). Expression of sphr-1 in RIC fully rescued RIC activities, yet in contrast to the sphk-1 mutant, S1P failed to restore RIC responses of the sphr-1 mutant (Figures 5E, 5F, and S6F). We conclude that in response to mitochondrial stress in non-neural tissues, S1P remodels RIC response properties via the SPHR-1 receptor.

Figure 5. — Time functions (A, C, and E) and area under curve (AUC) (B, D, and F) of calcium responses in the distal RIC axon evoked by the OP50 supernatant. The yellow shading (A, C, and E) represents stimulation by the OP50 cues. Results are mean ± SEM, and p values are indicated. N = neurons recorded. One-way ANOVA followed by Tukey’s correction (B, D, and F). See also Figure S6.

Sphingolipid signaling promotes avoidance of the pathogen C. indologenes

To explore whether sphingolipid signaling is also required for learned aversion of pathogens, we examined P. aeruginosa and C. indologenes, two C. elegans pathogens that induce mitochondrial stress.^14,45 Consistent with prior studies, adult C. elegans developed progressive P. aeruginosa avoidance after 4–6 h of exposure (Figure S7A). The sphk-1 mutant showed comparable P. aeruginosa avoidance with that of the wild type at either 6 or 15 h after exposure (Figure S7B). Moreover, antimycin treatment did not increase daf-7/transforming growth factor β (TGF-β) expression in the ASI and ASJ sensory neurons, a signature of P. aeruginosa infection.⁴⁶ These observations suggest that sphk-1 signaling is not involved in the learned aversion for P. aeruginosa.

Interestingly, adult C. elegans developed avoidance behavior after 3–6 h on C. indologenes (Figure 6A), which reached nearly 100% by 24 h (Figure 6B). In the chemotaxis assay, naive worms were attracted to C. indologenes, while worms cultivated on C. indologenes for 15 h avoided it (Figure 6C), suggesting that worms develop learned aversion for C. indologenes. Strikingly, avoidance of C. indologenes was completely abolished in the sphk-1 mutant even with intact locomotion on this pathogen (Figures 6A, 6B, and S7C–S7E), which could be partially rescued by S1P (Figure 6D). The spl-2 mutation, which is expected to increase S1P levels, enhanced C. indologenes avoidance at 6 h (Figure 6E). Moreover, expression of sphk-1 in the intestine or hypodermis, but not in neurons, significantly rescued the sphk-1 mutant for C. indologenes avoidance (Figure 6F). We found that a mutation of tbh-1/tyramine β-hydroxylase, which is required for octopamine synthesis in RIC, mildly reduced C. indologenes aversion (Figure 6G), implicating RIC neurons in the avoidance behavior for this pathogen. To our surprise, Chryseobacterium supernatant induced RIC responses in a fraction of naive worms, but this innate RIC response to Chryseobacterium cues was reduced in worms that were cultivated on C. indologenes for 15 h (Figures 6H and 6I). By contrast, there was no innate RIC response to OP50 E. coli cues, unless the worms were primed with mitochondrial stress (Figure 5). These findings imply that while aversive learning under mitochondrial stress and Chryseobacterium infection shares common signaling mechanisms (sphingolipids) and neuronal elements (RIC), each also engages mechanisms specified by distinct stressors. It is possible that under Chryseobacterium infection, S1P also targets other neurons that in turn modulate RIC activity.

DISCUSSION

Metabolic states regulate learning and behavior, but the identity and mechanisms of the relevant signaling molecules are largely elusive. Using C. elegans, we show that intercellular sphingolipid signaling regulates aversive learning under mitochondrial stress (Figure 7). S1P is sufficient to restore aversive learning in the sphk-1 mutant, and it acts upon the SPHR-1 receptor in the octopaminergic RIC neuron. Importantly, this sphingolipid pathway promotes learned aversion of a natural C. elegans pathogen. Such a gut/hypodermal-brain signaling axis serves as a paradigm for metabolic regulation of state-dependent behavioral plasticity.

Figure 7. — S1P produced from the intestine and hypodermis under mitochondrial stress or pathogen infection promotes aversive learning by targeting the RIC neuron, completing a gut/hypodermis-brain signaling circuit.

Sphingolipid signaling encodes an internal state of mitochondrial stress

Signaling molecules derived from the digestive tract regulate feeding behavior. In mammals, ghrelin from the gastrointestinal tract signals hunger to hypothalamic neuropeptide Y (NPY) neurons to promote feeding.^47–49 In C. elegans, feeding behavior is controlled in part by gut bacteria-derived tyramine, which modulates food preference and chemotactic behaviors.⁵⁰ Infection and allergy trigger host avoidance of pathogens or allergens in a process that involves neuropeptides, lipid metabolites, or damage-associated molecular patterns (DAMPs).^7,8,46,51,52 Small peptides, such as cytokines, released from injured tissues can modulate behavior by altering synaptic transmission or gene expression.⁵³ Tissue inflammation also impacts sphingolipid metabolic pathways,⁵⁴ and sphingolipids, including S1P, are important regulators of mitochondrial homeostasis and oxidative stress responses in C. elegans.^14,16,55 In addition to the intestine, data here and from our recent study also implicate hypodermis as an important tissue that signals to the brain for aversive learning.⁵⁶ C. elegans hypodermis can function as an endocrine organ that accumulates lipids⁵⁷ and that secretes signals to regulate developmental diapause, protein homeostasis, and metabolism.^58,59 In addition to its known functions in patterning the excretory cells and the apical extracellular matrix during larval development,³⁶ we show that the lipocalin LPR-3 is also an S1P chaperone. Expression of lpr-3 is undetectable in adults without stress but is markedly increased by mitochondrial dysfunction, suggesting that LPR-3 has a conditional role in responding to tissue stress. Importantly, S1P does not trigger avoidance behavior in the absence of stress, suggesting that other stress-associated factors are required. As S1P is also involved in physiological functions unrelated to stress, pairing S1P with other stress-related factors ensures the specificity of behavioral modulation and prevents inadvertent induction of unwanted avoidance behaviors under non-stressful conditions.

Sphingolipid signaling promotes learned pathogen avoidance

Among the five mammalian S1PRs,⁶⁰ neuronal S1PR2 mediates Nogo-A-dependent synaptic depression,⁶¹ and S1PR3 in the retina controls migration of the Müller glial cells.⁶² We show that S1P signaling modulates neuronal functions to regulate aversive learning in C. elegans. As SPHR-1 is a metabotropic receptor, we speculate that SPHR-1 regulates RIC responses via second messengers, such as diacylglycerol and inositol triphosphate, which modulate ion channels directly or indirectly via calcium released from the endoplasmic reticulum (ER).⁶³ The importance of SPHK-1 and S1P in learned avoidance of C. indologenes suggests that sphingolipid signaling is an innate mechanism for learned pathogen avoidance. Interestingly, SPHK-1 is dispensable for learned avoidance of P. aeruginosa, another C. elegans pathogen. Differential requirement of sphingolipid signaling for aversive learning of different pathogens suggests that C. elegans interprets specific metabolic codes post-infection to distinguish various pathogens in its behavioral responses. Pseudomonas and Chryseobacterium spp. produce siderophores, iron-chelating compounds that potentially damage iron-rich mitochondria. Pyoverdins and pyochelin, two siderophores produced by P. aeruginosa, activate daf-7/TGF-β expression in C. elegans ASJ sensory neurons, which promotes Pseudomonas avoidance.⁴⁶ Chryseochelins, citrate-based siderophores produced by Chryseobacterium spp., differ significantly from pyochelin and pyoverdin in chemical structures.⁶⁴ Chryseochelins are implicated in plant defense against infections, such as Ralstonia solanaceanum, a tomato pathogen.⁶⁴ The molecular basis of Chryseobacterium toxicity in animal cells remains unknown. We speculate that different siderophores impair mitochondria through distinct mechanisms, such as target molecule specificity. Why does C. elegans employ diverse mechanisms for learned aversion of different pathogens? Although both Pseudomonas and Chryseobacterium are found in natural habitats of C. elegans, Pseudomonas spp. are anaerobic enterobacteriae, whereas Chryseobacterium spp. are aerobic, rod-like pathogens. This suggests that they occupy different niches of distinct environmental conditions, such as oxygen concentration and temperature. It could be beneficial for C. elegans to distinguish pathogens, as each pathogen is likely associated with a distinct environmental context. As naive C. elegans shows preference for Pseudomonas and Chryseobacterium, preservation of attraction to pathogens through evolution suggests that the value of these pathogens is conditional on the physiological and ecological contexts of exposure. In nature, animals are exposed to myriad pathogens and stressors, each of which may alter behavior through unique combinations of life history, sensory cues, and non-neural signals, enabling complex and conditional behavioral plasticity.

RESOURCE AVAILABILITY

Lead contact

Requests for strains, reagents, and protocols should be directed to and will be fulfilled by the lead contact, Chun-Liang Pan (chunliangpan@gmail.com).

Materials availability

C. elegans strains and DNA constructs generated in this study are available from the lead contact upon request. The CHO-K1 cell lines stably expressing mitochondrial-targeted apo-aequorin are under a material transfer agreement and cannot be freely distributed.

Data and code availability

All the primary data for this study have been deposited at Figshare and are publicly available as of the date of publication, with the DOI in the key resources table.
This study does not use original code for data analysis.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

KEY RESOURCES TABLE.

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
GFP Antibody (B-2)	Santa Cruz Biotechnology	sc-9996
beta actin Antibody (C4)	Santa Cruz Biotechnology	sc-47778
beta tubulin Antibody (2G7D4)	GenScript	A01717
HRP Goat anti-mouse IgG Antibody	BioLegend	405306
GFP-Trap^® Agarose	ChromoTek	AB_2631357
Bacterial and virus strains
E. coli: Strain OP50	Caenorhabditis Genetics Center (CGC)	WormBase: OP50
E. coli: Strain HT115	Caenorhabditis Genetics Center (CGC)	N/A
P. aeruginosa: Strain PA14		N/A
Chryseobacterium indologenes	ATCC	29897
Chemicals, peptides, and recombinant proteins
Antimycin A	Sigma-Aldrich	A8674
IPTG (isopropyl b-D-1-thiogalactopyranoside)	Sigma-Aldrich	I6758
GENEzol	Geneaid	GZX100/D100
Sphingosine-1-phosphate (d18:1)	Cayman Chemical	62570
Benzaldehyde	Sigma-Aldrich	B1334
1-Octanol	Alfa Aesar	A15977
2-Nonanone	Sigma-Aldrich	108731
C16 Ceramide (d18:1/16:0)	Cayman Chemical	10681
S1P-biotin	Echelon Bioscience	S-200B
Streptavidin-HRP conjugate	Invitrogen^™	S911
Deposited data
Raw data of behavior assays	This paper	https://doi.org/10.6084/m9.figshare.24316951.v1
Experimental models: Cell lines
Hamster: CHO cells	ATCC	CCL-61
Xenopus laevis oocytes	Ecocyte Bio Science	N/A
Experimental models: Organisms/strains
C. elegans: Strain N2: wild isolate	CGC	WormBase: N2
C. elegans: Strain VC916: sphk-1(ok1097)/II	CGC	WormBase: sphk-1
C. elegans: Strain RB1465: C23H3.4(ok1693)/II	CGC	WormBase: sptl-1
C. elegans: Strain VC2358: sptl-2(ok2753)/V	CGC	WormBase: sptl-2
C. elegans: Strain VC747: lagr-1(gk327)/I	CGC	WormBase: lagr-1
C. elegans: Strain RB1036: hyl-1(ok976)/IV	CGC	WormBase: hyl-1
C. elegans: Strain JCM1: hyl-2(gnv-2)/X	CGC	WormBase: hyl-2
C. elegans: Strain RB1498: hyl-2(ok1766)/X	CGC	WormBase: hyl-2
C. elegans: Strain VC242: spl-2(ok490)/V	CGC	WormBase: spl-2
C. elegans: Strain CLP1511: sphk-1(twn21) linker gfp/II; twnEx704(Pelt-2::tomm20::mCherry, Pgcy-8::mCherry)	This Paper	N/A
C. elegans: Strain CLP1213: sphk-1(ok1097)/II; twnEx525(Pdpy-7::sphk-1, Pdpy-7::gfp)	This Paper	N/A
C. elegans: Strain CLP1214: sphk-1(ok1097)/II; twnEx526(Pelt-2::sphk-1, Pelt-2::gfp)	This Paper	N/A
C. elegans: Strain CLP1395: sphk-1(ok1097)/II; twnEx633(Punc-119::sphk-1, Pgcy-8::gfp)	This Paper	N/A
C. elegans: Strain CLP1494: sphk-1(ok1097)/II; twnEx681(Pelt-2::sphk-1(Δcam), Pelt-2::gfp)	This Paper	N/A
C. elegans: Strain CLP1495: sphk-1(ok1097)/II; twnEx682(Pelt-2::sphk-1(KD), Pelt-2::gfp)	This Paper	N/A
C. elegans: Strain UP3047: csEx603(Plpr-3::ssSfGFP::lpr-3, Plin-48::mRFP)	Meera Sundaram’s Lab	N/A
C. elegans: Strain CLP1178: twnEx509(Pelt-2::Cas9, PU6::lpr-3 sgRNA, Pelt-2::gfp)	This Paper	N/A
C. elegans: Strain CLP1211: twnEx523(Pdpy-7::Cas9, PU6::lpr-3 sgRNA, Pdpy-7::gfp)	This Paper	N/A
C. elegans: Strain CLP1398: sphk-1(1097)/II; twnEx509(Pelt-2::Cas9, PU6::lpr-3 sgRNA, Pelt-2::gfp)	This Paper	N/A
C. elegans: Strain CLP1399: sphk-1(1097)/II; twnEx523(Pdpy-7::Cas9, PU6::lpr-3 sgRNA, Pdpy-7::gfp)	This Paper	N/A
C. elegans: Strain CLP1469: srsx-22(twn20)/V	This Paper	N/A
C. elegans: Strain CLP1294: F10D7.1(twn15)/X	This Paper	N/A
C. elegans: Strain FX04341: F57A8.4(tm4341)/V	NBRP	WormBase: npr-39
C. elegans: Strain CLP1400: sphr-1(twn18)/V	This Paper	N/A
C. elegans: Strain CLP1512: twnEx705(Psphr-1::mCherry, Ptbh-1::Neongreen, Punc-17::bfp)	This Paper	N/A
C. elegans: Strain CLP1426: sphr-1(twn18)/V; twnEx642(Psphr-1::sphr-1, Psphr-1::mCherry)	This Paper	N/A
C. elegans: Strain CLP1427: sphr-1(twn18)/V; twnEx643(Punc-17::sphr-1, Psphr-1::mCherry)	This Paper	N/A
C. elegans: Strain CLP1428: sphr-1(twn18)/V; twnEx644(Ptbh-1::sphr-1, Psphr-1::mCherry, Ptbh-1::t2a::NG)	This Paper	N/A
C. elegans: Strain CLP1418: sphk-1(ok1097)/II; sphr-1(twn18)/V	This Paper	N/A
C. elegans: Strain CLP1474: sphr-1(twn18)/V; twnEx673(Ptbh-1::hS1PR2, Pgcy-8::mCherry)	This Paper	N/A
C. elegans: Strain CLP1475: sphr-1(twn18)/V; twnEx674(Ptbh-1::hS1PR3, Pgcy-8::mCherry)	This Paper	N/A
C. elegans: Strain CLP1656: sphr-1(twn18)/V; twnEx804(Ptbh-1::SPHR-1(4A), Pgcy-8::gfp)	This Paper	N/A
C. elegans: Strain CLP1251: twnEx557(Ptbh-1::mcasp1, Podr-1::gfp)	This Paper	N/A
C. elegans: Strain CLP1396: sphk-1(ok1097)/II; twnEx557(Ptbh-1::mcasp1, Podr-1::gfp)	This Paper	N/A
C. elegans: Strain CLP1425: sphr-1(twn18)/V; twnEx557(Ptbh-1::mcasp1, Podr-1::gfp)	This Paper	N/A
C. elegans: Strain FX17797: ser-6(tm2146)/IV	NBRP	WormBase: ser-6
C. elegans: CLP1577: ser-6(tm2146)/IV; sphr-1(twn18)/V	This Paper	N/A
C. elegans: Strain CLP1335: twnEx608(Ptbh-1::GCaMP6s, Ptdc-1::bfp)	This Paper	N/A
C. elegans: Strain CLP1397: sphk-1(ok1097)/II; twnEx608(Ptbh-1::GCaMP6s, Ptdc-1::bfp)	This Paper	N/A
C. elegans: Strain CLP1429: sphr-1(twn18)/V; twnEx608(Ptbh-1::GCaMP6s, Ptdc-1::bfp)	This Paper	N/A
C. elegans: Strain CLP1501: sphr-1(twn18)/V; twnEx608(Ptbh-1::GCaMP6s, Ptdc-1::bfp), twnEx684(Ptdc-1::sphr-1::SL2::bfp)	This Paper	N/A
C. elegans: Strain CLP1306: twnEx766(Pelt-2::gfp)	This Paper	N/A
C. elegans: Strain FK181: ksIs2(Pdaf-7::GFP, rol-6(su1006))	CGC	WormBase: ksIs2
C. elegans: Strain CLP1554; ksIs2(Pdaf-7::GFP, rol-6(su1006)); twnEx733(Pstr-3::mCherry, Punc-17::bfp)	This Paper	N/A
C. elegans: Strain MT9455: tbh-1(n3247)/X	CGC	WormBase: tbh-1
Oligonucleotides
smFISH probes for lpr-3, see Table S1	LGC Biosearch Technologies	N/A
sgRNA sequence: lpr-3: ACGACGTTAGCTGTGGCACT	This Paper	N/A
sgRNA sequence: sphr-1: TCTCGACAACATGTCATCGG	This Paper	N/A
sgRNA sequence: srsx-22: AGTATCAAACGAAACAATGG	This Paper	N/A
sgRNA sequence: F10D7.1: GTACACATTGACCAGTAACG	This Paper	N/A
crRNA sequence: sphk-1: CTAGGCAGTTGATGAGAAAA	This Paper	N/A
Primers for Recombinant DNA and genotyping, see Table S1	This Paper	N/A
Recombinant DNA
Pelt-2::tomm20::mCherry	This paper	N/A
Pgcy-8::mCherry	This paper	N/A
Pdpy-7::sphk-1	This paper	N/A
Pdpy-7::gfp	This paper	N/A
Pelt-2::sphk-1	This paper	N/A
Pelt-2::gfp	This paper	N/A
Punc-119::sphk-1	This paper	N/A
Pgcy-8::gfp	This paper	N/A
Pelt-2::sphk-1(Δcam)	This paper	N/A
Pelt-2::sphk-1(KD)	This paper	N/A
Pelt-2::Cas9	This paper	N/A
PU6::lpr-3 sgRNA	This paper	N/A
Pdpy-7::Cas9	This paper	N/A
Psphr-1::mCherry	This paper	N/A
Ptbh-1::Neongreen	This paper	N/A
Punc-17::bfp	This paper	N/A
Psphr-1::sphr-1	This paper	N/A
Punc-17::sphr-1	This paper	N/A
Ptbh-1::sphr-1	This paper	N/A
Ptbh-1::hS1PR2	This paper	N/A
Ptbh-1::hS1PR3	This paper	N/A
Ptbh-1::sphr-1(alkyl mutant)	This paper	N/A
Ptbh-1::mcasp1	This paper	N/A
Podr-1::gfp	This paper	N/A
Ptbh-1::GCaMP6s	This paper	N/A
Ptdc-1::bfp	This paper	N/A
Ptdc-1::sphr-1::SL2::bfp	This paper	N/A
Pstr-3::mCherry	This paper	N/A
Software and algorithms
ImageJ	NIH	https://imagej.nih.gov/ij/
WormLab	MBF Bioscience	https://www.mbfbioscience.com/
The PyMOL Molecular Graphics System	Schriödinger, LLC	https://pymol.org/2/
Metaboseek	Cornell University	https://metaboseek.com/
Xcalibur QualBrowser v4.1.31.9	Thermo Fisher Scientific	https://www.thermofisher.com/
SPARKCONTROL	TECAN	https://www.tecan.com/
Roboocyte2+	Multi Channel Systems MCS GmbH	https://www.multichannelsystems.com/
Prism 10	Graphpad	https://www.graphpad.com/
AlphaFold2	Google DeepMind	https://deepmind.google/technologies/alphafold/
ZEN Microscopy Software	Zeiss	https://www.zeiss.com/microscopy/en/home.html
Metamorph	Molecular Devices	https://www.moleculardevices.com

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STAR★METHODS

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Animals

C. elegans strains were cultured and maintained as previously described.⁶⁵ Animals are maintained on nematode growth medium (NGM) agar plates seeded with E. coli OP50 bacterial strain as food at 20°C unless specified otherwise. Young (D1) adult hermaphrodite animals were used for all behavioral experiments. For genetic crosses, genotypes were confirmed by sequencing or PCR. A list of mutant alleles and transgenic strains used in this study is available in key resources table.

Microbe Strains

The OP50 and HT115 E. coli strains are used.

Cell Lines

CHO-K1 cells stably expressing mitochondrial-targeted apo-aequorin and a promiscuous human Gα₁₆ protein were used for GPCR activation assays (ES-000-A24, PerkinElmer). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/Nutrient Mixture F-12 Ham (Sigma-Aldrich), supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 1% penicillin/streptomycin mixture (10.000 units/ml penicillin and 10 mg/mL streptomycin, Gibco), and 250 mg/mL zeocin. Cells were grown in stable conditions of 37 °C, 5% CO2 and high relative humidity. Mycoplasma tests were performed using the MycoAlert Mycoplasma Detection Kit (Lonza) to verify that cells were free of mycoplasma contamination.

METHOD DETAILS

Molecular Biology and Germline Transformation

Promoters used in this study included those for dpy-7 (0.2 kb), elt-2 (3.9 kb), tbh-1 (4.5 kb), tdc-1 (4.6 kb), unc-17 (3.2 kb), unc-119 (1.7 kb) and sphr-1 (6 kb). These were cloned into pPD95.75 or pPD95.77 vectors. Genomic DNA for sphk-1 (2.3 kb), and cDNA for sphr-1 (1.3 kb), human S1PR2 (1 kb) and human S1PR3 (1.1 kb) were used in respective experiments. Germline transformation by microinjection was performed as described.⁶⁶ Transgenic animals were generated by injecting DNA plasmids with fluorescent co-injection marker into young adult hermaphrodites using micropipette glass capillary or CRISPR/Cas9 genome editing. Primer information is available in Table S1.

CRISPR/Cas9 Genome Editing

For generating mutants, the constructs pDD162(Peft-3::Cas9, Addgene #47549) and sgRNAs (C24B5.1: TCTCGACAACATGTCATCGG; srsx-22: AGTATCAAACGAAACAATGG; F10D7.1: GTACACATTGACCAGTAACG) were injected in young adult hermaphrodites by germline transformation. An unc-22 sgRNA construct was co-injected as a co-CRISPR marker to select F1transgenic animals. Animals with twitcher phenotypes indicate possible editing at the targeted gene locus. F1 animals were lysed for targeted gene PCR products followed by performing T7-endonuclease I digestion.^37,67 The mutation sites were confirmed by sequencing. For generating knock-in GFP strains, CRISPR/Cas9 genome editing was performed as described with some modifications.²⁹ sphk-1 crRNA (CTAGGCAGTTGATGAGAAAA) (Integrated DNA Technologies (IDT), Coralville, Iowa, USA). To make GFP flexible and not affect the protein function, we added GS linker (90 bp) in between sphk-1 stop codon and GFP.

Preparation of the dsDNA asymmetric donors

140 bp (120 bp homology arms and 20 bp complementary to insertion) oligos and standard oligos complementary to insertion site sequences were ordered from IDT. 2 μg of each PCR products (~1.1 kb and 0.9 kb) were mixed and heated to 95°C followed by cooling to 4°C for re-annealing.

Preparation of the injection mixtures

0.5 μL of Cas9 (10 μg/μL), 0.5 μL tracrRNA and 0.5 μL crRNA (2 nmol, all from IDT) were mixed together gently and incubated at 37 °C for 15 minutes for RNP complex formation. The dsDNA donor cocktail was added (total 4 μg) in the final injection mixtures. pRF4(rol-6 (su1006)) (40 ng/μL) was co-injected as a co-injection marker. Nuclease-free water was added to a total volume of 20 μL. The final injection mixture was centrifuged at 14000 rpm for 2 minutes and injected into young adult hermaphrodites. Successful insertion of fluorescent tags was screened using a fluorescence dissecting microscope and confirmed by PCR and sequencing.

Bacterial Avoidance Assay with Antimycin A

Bacterial avoidance assay was performed as reported.^23,25 OP50 was cultured in lysogeny broth (LB) at a density of O.D. 0.4–0.6. One hundred microliters (100 μL) of OP50 was seeded at the center of the 5.5 cm NGM plate the day before the bacterial avoidance assay. Antimycin A (Sigma-Aldrich) from the stock (4.5 mM in absolute EtOH) was diluted with M9 buffer and added in the OP50 plates to final concentration 4.5 μM or other specific concentration as indicated. For the control group, an equal amount absolute EtOH was diluted and added in the OP50 plates. Young adult/D1 animals were used in bacterial avoidance assay. To prepare synchronized animals, five gravid hermaphrodites were placed to lay eggs on NGM plates seeded with OP50 for 6–8 hours. The adults were removed and the progenies were grown 72 hours to reach young adult/D1. Young adult/D1 animals were collected by washing with M9 for three times and settled to the bottom of the tube by gravity. The worms were released on the OP50 plates supplemented with Antimycin A or control for six hours.

Avoidance Assay with RNAi

RNAi avoidance assays were performed as previously described with some modifications.⁵ The RNAi bacterial culture (empty vector, spl-1, spl-2) was diluted at 1:100 ratio and grown at 37°C to OD. 0.4–0.6, followed by 1mM isopropyl b-D-1-thiogalactopyranoside (IPTG) induction for 1 h. One hundred microliters (150 μL) of HT115 was seeded at the center of the 5.5 cm NGM plate. The seeded plate was stand at room temperature overnight for inducing double-stranded RNA. Adult animals were transferred on the plate for laying eggs. After eggs hatched and grew to young adult, these animals were washed and performed bacterial avoidance assay with antimycin A as previously described.

Bacterial Chemotaxis Assay

Bacterial chemotaxis assays were performed as previously described.^23,25 The 5.5 cm chemotaxis (CTX) plates (2% agar, 5 mM potassium phosphate, pH 6.0, 1 mM CaCl₂, 1 mM MgSO₄) was divided into six equal longitudinal zones similar to that used for repulsive chemotaxis assays. 1 μL of OP50 liquid culture and 1 μL control medium (LB) were spotted on the opposite site of the plate. 1 μL of 0.5 M sodium azide was placed on the both sites to immobilize the worms once they reach the odorant or control spots. Young adult/D1 animals were trained on E. coli OP50 plates with 1.125 μM antimycin A or control for three hours. The animals were washed with M9 buffer twice and transferred to NGM plates seeded with E. coli HT115 for 15 hours. On the second day, the animals were washed with CTX buffer (5 mM potassium phosphate (pH 6.0), 1 mM CaCl₂, 1 mM MgSO₄) twice and once with water. Animals were transferred to the center of the CTX plates. Two hours later, the chemotaxis index (CI) was quantified as

C I = N_{O P 50} - N_{L B} / N_{O P 50} + N_{L B}

where $N_{O P 50}$ is the number of worms in the two zones close to OP50, and $N_{L B}$ is the number of worms in the two zones close to LB.

Odorant Chemotaxis Assay

Odorant chemotaxis was performed as described with several modifications. For the attractive odor benzaldehyde (1:200, Sigma-Aldrich, B1334), the 10 cm chemotaxis plate (2% agar, 5 mM potassium phosphate, pH 6.0, 1 mM CaCl₂, 1 mM MgSO₄) were divided into four quadrants. A 2 cm circle centered at the odorant/ control spot was drawn on the plate. 1 μL of diluted benzaldehyde or control was spotted on the opposite site of the plate. 1 μL of 0.5 M sodium azide was placed on the both sites to immobilize the worms once they reach the odorant or control spots. Young adult or D1 animals were collected from the NGM plates and washed twice with CTX buffer and once with water. Animals were released on the origin quadrant (between the test odor and the control) and moved for an hour. The chemotaxis index is calculated as

C h e m o t a x i s i n d e x (C I) = (N o d o r - N c o n t r o l) / N t o t a l

where Nodor and Ncontrol are numbers of animals in the circles of odor or control, respectively.

For the repulsive odor 1-octanol (undiluted, Alfa Aesar, A15977) or 2-nonanone (1:10, Sigma-Aldrich, 108731), the 10 cm chemotaxis plate (2% agar, 5 mM potassium phosphate, pH 6.0, 1 mM CaCl₂, 1 mM MgSO₄) were divided into six longitudinal zones. Two spots of 1 μL 1-octanol/ 1 μL of diluted 2-nonanone or control solvent were spotted on the opposite site of the plate. 1 μL of 0.5 M sodium azide was placed on the both sites to immobilize the worms once they reach the odorant or control spots. Young adult or D1 animals were collected from the NGM plates and washed twice with CTX buffer and once with water. Animals were released on the middle zone of the plate and allowed to move for an hour. The chemotaxis index is calculated as

C h e m o t a x i s i n d e x (C I) = (N o d o r - N c o n t r o l) / N t o t a l

where Nodor and Ncontrol are numbers of animals in the zones of odor or control, respectively.

S1P and Ceramide Supplement

Sphingosine-1-phosphate (d18:1) and ceramide (d18:1/16:0) were dissolved in methanol and supplemented to NGM plates to reach the final concentration of 10 μM. The plates were allowed to sit overnight before use. For bacterial chemotaxis, young adult animals were transferred to the S1P (d18:1) plates for three hours before training or testing.

Metabolomic analysis of worms using HPLC-HRMS

Egg Preparation

Wild-type or mutant worms on 9 cm NGM plates seeded with E. coli OP50 were cultured at 22°C. The worms were cultured till the food was consumed and the eggs hatched. Worms of three 9 cm NGM plates were transferred in 25 mL S-complete medium in a 250 mL Erlenmeyer flask. Three grams (3 g) of E. coli OP50 grown to stationary phase in Terrific Broth was supplemented in the liquid culture. The liquid culture was shaken at 120 RPM under 22°C for 72 h. The gravid animals were collected by centrifugation at 3000 g for 1 min and discard the supernatant. 20 mL of freshly prepared alkaline-bleach solution (15 mL of water, 4 mL of commercial bleach, and 1 mL 10N NaOH) was added to the worm pellet and shake for 5–10 minutes to collect eggs. Eggs were washed for twice, resuspended in 20 mL M9 buffer, and rotated on a rocker for 24 h at 22°C.

Worm Culture

After worms hatched, 700,000 L1 larvae were counted and seed in the 45 mL of S-complete medium in a 250 mL Erlenmeyer flask. 6 g of E. coli OP50 was supplemented in the liquid culture which was shaken at 120 RPM under 22°C for 48 h, when the worms reached young adult. Antimycin A or absolute ethanol were added in the liquid culture to final concentration 4.5 μM, respectively for three hours mitochondrial stress. Worms were pelleted at 3000 g for 1 minute. The liquid culture media contains exo-metabolome and the worm pellet contains endo-metabolome. The worm pellet was washed twice with 30 mL of S-complete medium and transferred to a new 250 mL Erlenmeyer flask in 30 mL of S-complete medium and 1.5 g of E. coli HT115 to recover from stress for 3 h. Worms were pelleted at 3000 g for 1 minute and exo-and endo-metabolomes were collected as described above. The liquid culture media and worm pellet were frozen in −80°C for further metabolomic analysis. Three biological replicates were grown for all genotype/conditions.

Metabolomics Sample Preparation

Worm pellet (endo-metabolome) samples were lyophilized for 24 h using a VirTis BenchTop 4 K Freeze Dryer. After the addition of 1 mL methanol directly to the conical tube in which animals were frozen, samples were sonicated for 5 min (2 s on/off pulse cycle at 90 A) using a Qsonica Q700 Ultrasonic Processor with a water bath cup horn adaptor (Qsonica 431C2). Following sonication, 2 mL of methanol was added and tubes were gently rocked overnight at room temperature. The conical tubes were centrifuged (2,750 × g, 20°C, 5 min) and the clarified supernatant was transferred to a clean 8 mL glass vial for further concentration to dryness in an SC250EXP Speedvac Concentrator coupled to an RVT5105 Refrigerated Vapor Trap (Thermo Scientific). The resulting powder was suspended in 50 μL of methanol, vortexed for 30 s and sonicated for 5 min. The suspensions were transferred to 1.7 mL Eppendorf tubes and centrifuged (18,000 X g, 22°C, 10 min). The resulting clarified supernatant was transferred to HPLC vials and analyzed directly by HPLC-HRMS.

HPLC Coupled with HRMS

Liquid chromatography was performed on a Vanquish HPLC system controlled by Chromeleon Software (ThermoFisher Scientific) and coupled to an Orbitrap Q-Exactive High Field mass spectrometer controlled by Xcalibur software (ThermoFisher Scientific). 2 μL of the methanolic extract prepared as described above was injected and separated on a Thermo Hypersil Gold C18 column (150 mm × 2.1 mm, particle size 1.9 μM, part no. 25002–152130) maintained at 40°C with a flow rate of 0.5 mL/min. Solvent A: 0.1% formic acid (Fisher Chemical Optima LC/MS grade; A11750) in water (Fisher Chemical Optima LC/MS grade; W6–4); solvent B: 0.1% formic acid in acetonitrile (Fisher Chemical Optima LC/MS grade; A955–4). A/B gradient started at 1% B for 3 min after injection and increased linearly to 98% B at 20 min, followed by 5 min at 98% B, then back to 1% B over.1 min and finally held at 1% B for the remaining 2.9 min to re-equilibrate the column (28 min total method time).

Mass spectrometer parameters: spray voltage, −3.0 kV/+3.5 kV; capillary temperature 380°C; probe heater temperature 400°C; sheath, auxiliary, and sweep gas, 60, 20, and 2 AU, respectively; S-Lens RF level, 50; resolution, 120,000 at m/z 200; AGC target, 3E6. Each sample was analyzed in negative (ESI−) and positive (ESI+) electrospray ionization modes with m/z range 100–1000.

HPLC- high-resolution mass spectrometry (HRMS) RAW data were converted to mzXML file format using MSConvert (v3.0, ProteoWizard) and were analyzed using Metaboseek software (v0.9.9.4) with the following settings: 5 ppm, 2_20 peakwidth, 3 snthresh, 3_100 prefilter, FALSE fitgauss, 1 integrate, TRUE firstBaselineCheck, 0 noise, wMean mzCenterFun, −0.005 mzdiff. Default settings for XCMS feature grouping: 0.2 minfrac, 2 bw, 0.002 mzwid, 500 max, 1 minsamp, FALSE usegroups. Metaboseek peak filling used the following settings: 5 ppm_m, 3 rtw, TRUE rtrange, FALSE areaMode. Quantification was performed with Metaboseek software⁶⁸ or via manual integration using Xcalibur QualBrowser v4.1.31.9 (Thermo Fisher Scientific) using a 5-ppm window around the m/z of interest.

smFISH

The single-molecule fluorescence in situ hybridization (smFISH) experiments were performed as described.⁶⁹ All probes for hybridization were coupled to TAMRA-C9. Briefly, synchronized animals were cultured on NGM plates with OP50 at 20°C until L2 or L3. Animals were collected, washed by M9 and fixed with 4% paraformaldehyde in 1X PBS for 15 minutes (adjust the fixation time by each probe). After washed twice with 1X PBS, fixed worms were processed in 70% ethanol at 4°C for overnight. Hybridization was performed with 20% formamide overnight at 30°C in dark. After washed at least three times with wash buffer and 30 minutes interval, animals were then mounted with 2XSSC and ProLong Gold Antifade Reagent (Invitrogen) and counter-stained with DAPI for image analysis. Confocal z-stack series were taken, with fluorescent marker labeled intestinal or hypodermal cells. To quantify smFISH signals in the intestine, we counted the number of RNA granules (> 0.2 μm) in area of 50–200 μm² (with 1–4 intestinal cells) and calculated the average puncta number per 100 μm². For smFISH signals in the hypodermis, we counted the number of RNA granules (> 0.5 μm in area of 500–1300 μm², with 7–10 hypodermal cells) and calculated the average puncta number per 100 μm². Information of the oligos is available in Table S1.

S1P Binding Assay

We used the transgenic strain UP3047: csEx603(Plpr-3::sssfGFP::LPR-3, Plin-48::mRFP) and a strain expressing GFP in the intestine as control. Animals were cultivated on 9 cm NGM plates seeded with E. coli OP50 at 20°C till young adult. Animals from 30 such plates were collected by washing with M9 buffer for at least three time to remove the bacteria, followed by a wash with lysis buffer (20 mM Tris-HCl pH7.5, 100 mM NaCl, 0.05% Triton-X-100). The worm lysis buffer was supplemented with 1mM Na3VO4, 10mM NaF and protease inhibitor cocktail (Roche, 04693132001). The worm pellet (0.25 mL) was transferred into a screw cap tube (DOT Scientific) with 0.5 mm glass beads (0.5mL, BioSpec Products, 11079105) and lysis buffer containing protease inhibitor cocktails. Tubes were placed in FastPrep-24 5G Benchtop Homogenizer (MP Biomedicals) and homogenized with three 30 seconds pulses at maximal speed. Proteins were collected by centrifugation with 15500 g for 30 minutes to remove insoluble proteins. A total of 10 mg proteins with 30 μl of GFP nanobody (GFP-Trap, Chromotek) were used for perform the lipid binding assay. Lysates (8 mg) from worms expressing GFP::LPR-3 were precipitated by 8 μl of GFP nanobodies immobilized on beads. After removal of the supernatant, the precipitated GFP::LPR-3 proteins were then incubated with 8 μg biotin-conjugated S1P (Echelon Bioscience, S-200B). Unbound S1P-biotin was removed by washing. Streptavidin-HRP conjugates (Invitrogen^™, S911) were then added, followed by chemoluminescent measurement to quantify the level of S1P-biotin bound to GFP::LPR-3 using a plate reader (TECAN Spark Multimode Microplate Reader, software: SPARKCONTROL, setting: Luminescence, 500 ms, output: counts). In another set of experiments, unlabeled S1P (4 μg) was added to compete with S1P-biotin for GFP::LPR-3 binding. After washing and the addition of streptavidin-HRP conjugates, bound S1P-biotin levels were quantified by chemoluminescent measurement as above. As calibration, we performed western blot analysis for GFP and actin with respective antibodies (Santa Cruz Biotechnology, sc-9996, sc-47778) to calculate the GFP/actin ratio. The read counts of S1P-biotin were normalized against the GFP/actin ratio in respective experiment.

Calcium Mobilization Assay for SPHR-1 Activation by S1P

GPCR activation assays in cultured cells were performed as previously described with some modifications.⁴⁰ Chinese hamster ovary (CHO) K1 cells, stably overexpressing mitochondrial targeted apo-aequorin (mtAEQ) and the promiscuous G_α16 protein, were transiently transfected with pcDNA3.1-sphr-1 or empty pcDNA3.1 vector. S1P (d18:1) (Cayman Chemical, 62570) was tested in final concentrations from 10⁻⁵ to 10⁻¹⁴ M. Calcium responses were monitored as previously described on a MicroBeta2 LumiJET luminometer (PerkinElmer).⁴⁰ Concentration-response measurements were conducted in quadruplicate and in at least two independent experiments.

Receptor Expression in CHO/mtAEQ/Gα₁₆ Cells

To validate expression of SPHR-1 in CHO/mtAEQ/Gα₁₆ cells, sphr-1 cDNA was cloned into an egfp::pcDNA3.1 backbone using NEBuilder^® HiFi DNA Assembly kit. The transfection-grade plasmid sphr-1::gfp::pcDNA3.1 was obtained with the NucleoBond Xtra Maxi Plus kit. Prior to transfection, CHO/mtAEQ/Gα₁₆ cells (ES-000-A24, PerkinElmer) were seeded on 4-well chambered Borosilicate glass and grown to around 70% confluency. sphr-1::gfp::pcDNA3.1 plasmid was transfected into CHO/mtAEQ/Gα₁₆ cells with Invitrogen Lipofectamine LTX with Plus Reagent. After 24 h, the transfected cells were supplemented with fresh complete DMEM and transferred from 37°C to 28°C to improve receptor expression. At 48h post-transfection, GFP expression in cells was imaged using a ZEISS LSM900 Airyscan2 confocal microscope. The image in Figure S5C is one representative stack showing plasma membrane localization of SPHR-1. This experiment was repeated three times independently.

TEVC Recording

For expressing SPHR-1 in Xenopus laevis oocytes, sphr-1 cDNA was cloned into a KSM backbone using NEBuilder^® HiFi DNA Assembly kit. The obtained sphr-1::ksm plasmid was linearized by NotI-HF restriction enzyme (New England Biolabs) and purified by DNA Clean & Concentrator-5 kit (Zymo Research). Capped RNA was synthesized from the linearized sphr-1::ksm plasmid using mMESSAGE mMACHINE T3 Transcription kit (Thermofisher Scientific). The synthesized RNA was further purified by RNeasy Mini Kit (QIAGEN). Likewise, empty KSM plasmid, mGIRK1 and mGIRK2 (mouse G protein inward rectifying potassium channels) RNA were prepared as described above. Defolliculated Xenopus laevis oocytes (Ecocyte Bio Science) were kept in a V-shaped bottom 96-well plate with the physiological solution ND96 (96mM NaCl, 1 mM MgCl₂, 5 mM HEPES, 1.8 mM CaCl₂ 2H₂O, 2 mM KCl, pH 7.4 with NaOH). A mixture of 10 ng RNA (sphr-1 or empty KSM vector), 7.5 ng mGIRK1 RNA, and 7.5 ng mGIRK2 RNA was injected into individual oocytes using a Roboinject system (Multi Channel Systems MCS GmbH). After injection, oocytes were kept at 16°C for 48 h. On the day of recording, 1 μM S1P was freshly prepared by diluting 10mM S1P ready-made solution (MERCK) in high K⁺ solution (96 mM KCl, 1 mM MgCl₂, 5 mM HEPES, 1.8 mM CaCl₂, 2 mM NaCl). A two-electrode voltage clamp (TEVC) recording was conducted with a Roboocyte2 system (Multi Channel Systems MCS GmbH). Electrode heads (Multi Channel Systems MCS GmbH) were filled with 1.5M KAc and 1M acetic acid, with resistance ranging from 700–2200 μΩ. A customized script was run where oocytes are constantly clamped at −80mV and currents are measured during perfusion. Oocytes were initially perfused with ND96 solution for 10s, followed by 30s-40s perfusion of high K⁺ solution until the current reached a plateau to get the baseline opening of the GIRK channel. After the plateau, 80s-120s perfusion with 1μM S1P was applied to also reach a plateau where S1P elicits GIRK opening. Lastly, 30s perfusion of ND96 was applied to bring the oocyte to resting membrane potential. Data was processed using the Roboocyte2+ software, to export the minimum current value during the high K⁺ perfusion and S1P perfusion. The GIRK activation rate (%) was determined by calculating the ratio of the S1P-elicited minimum current and the baseline current elicited by high K⁺ solution. Graph plotting and statistical analysis were done using GraphPad Prism 10.

Epifluorescent and Confocal Microscopy

To quantify GFP intensity under mitochondrial stress, animals with csEx603(Plpr-3::ssSfGFP::lpr-3) were anesthetized with 1mM levamisole and 5 worms were aligned together. Worms are imaged by the Zeiss AxioImager2 microscope, and GFP pixel intensities were quantified by ImageJ after subtraction of background signals. To quantify SPHK-1 subcellular localization under mitochondrial stress, young adult SPHK-1 knocked-in GFP animals (sphk-1(twn21)) with mitochondrial labeled with mCherry were anesthetized with 1mM levamisole. Images were acquired by Zeiss LSM880 confocal microscope with Airyscan programs. All the animals were imaged for the posterior regions of the intestine. The images were processed by Airyscan joint deconvolution with selected iteration of SPHK-1 (iteration 10) and mitochondria (iteration 20). The colocalization coefficient was determined by the Zeiss Zen software.

Calcium Imaging in a Microfluidic Device

Calcium imaging using the microfluidic chip was performed as previously described with some modifications.⁴⁴ OP50 was cultured in 6 mL LB at 37°C for 8 h and centrifuged to discard the supernatant. The pellet was resuspended in 35 mL NGM buffer (with peptone to support bacterial growth) and cultured at 37°C for 12–16 h till O.D. reached 0.6. The bacterial culture was filtered by 0.22 μm filter membrane for use. Young adult or D1 worms trained with antimycin A or control were transferred into an olfactory microfluidic chip (MicroKosmos, Ann Arbor, MI, USA). NGM buffer or OP50 supernatant (O.D. around 0.6) was delivered through the VC-8 Valve Control System (Warner Instruments, Holliston, MA, USA) with journals written with MetaMorph (Molecular Devices, San Jose, CA, USA). Fluorescent images were acquired using the ORCA3 sCMOS CCD camera (Hamamatsu Photonics, Japan) and pE-300 LED (CoolLED, UK), with speed of 2 frames/s and 400 ms exposure time at 5% illumination intensity. Normalized fluorescence change (ΔF/F0) is calculated as where F₀ is the average of the fluorescent intensity five frames before giving stimulus (OP50 supernatant) for RIC neurons.

Locomotion Analysis

To quantify worm locomotion, well-fed young adult or D1 hermaphrodites were washed with M9 buffer to rid from bacteria. Around five worms were released on an unseeded NGM plates and recorded for three minutes using WormLab (MBF Bioscience, Vermont, USA). After manually correction of the automatic detection, locomotion parameters including speed, reversal frequency, and reversal times were analyzed.

Western Blotting

20 young adult animals were lysed in the lysis buffer with 95°C for five minutes and cooled on ice. The sample was mixed with sample buffer and load into the SDS-PAGE for electrophoresis followed by western blotting. We used the following primary antibodies: GFP Antibody (B-2) (1:1000, Santa Cruz Biotechnology, sc-9996), beta actin Antibody (C4) (1:2000, Santa Cruz Biotechnology, sc-47778), and beta tubulin Antibody (2G7D4) (1:2000, GenScript, A01717). The secondary antibody used in thus study was HRP Goat anti-mouse IgG Antibody (1:5000, BioLegend, 405306).

Modeling of Protein Structures by AlphaFold2

Structural prediction of proteins was performed as described.^70,71 The protein sequences were queried at Google ColabFold v1.5.2-patch: AlphaFold2 using MMseqs2 (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb). MMseqs2 generate diverse multiple sequence alignments (MSAs), from which the “unpaired_paired” mode is selected, which is pair sequences from same species plus unpaired MSA (separating MSA from each chain). Alphafold2_ptm is selected for the monomer prediction model. The number of recycles is 20. The recycle early stop tolerance is set to 0.5. The pairing strategy fits the greedy mode which is to pair any taxonomically matching subsets. The prediction model is labeled manually by PyMOL. Structural alignment between LPR-3 and human ApoM was also performed by PyMol, with cealign for the robustness of alignment due to low sequence similarity between the two proteins. The two aligned objects are: human ApoM (PDB: 2YG2) and C. elegans LPR-3 (ColabFold v1.5.2). The root-mean-square deviation (RMSD) is 4.145804 Å over 144 residues. S1P-interacting residues of LPR-3 are predicted by Autodock/Vina in PyMOL. Autodock/Vina docking predicts binding modes by evaluating the energetic favorability of various potential orientations within a protein’s binding pocket. The docking result can model the amino acids interacting with the polar head group of S1P and the hydrophobic alkyl chain. The S1P head group contains a negatively charged phosphate moiety. Positively charged and polar residues create favorable electrostatic environments. Hydrophobic residues stabilize the lipid through van der Waals interactions.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analyses used in this paper include two-way ANOVA with Bonferroni’s correction for multiple comparisons, Mann-Whitney U test, unpaired t-test, and extra sum-of-squares F test. For behavioral experiments such as the avoidance and bacterial chemotaxis assays, two-way ANOVA is used as we compare two categorical variables: the condition (EtOH, antimycin) and the genotype. Statistics were performed by Prism for experiments indicated in the Figure Legends. Error bars in the graphs represent standard error of means (S.E.M.).

Supplementary Material

NIHMS2130171-supplement-1.pdf^{(7.5MB, pdf)}

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.cub.2025.03.082.

Highlights.

SPHK-1 sphingosine kinase promotes aversive learning under mitochondrial stress
S1P acts through its G protein-coupled receptor SPHR-1
SPHR-1 remodels the properties of the octopaminergic neuron to promote avoidance
Sphingolipids promote learned aversion of the pathogen Chryseobacterium indologenes

ACKNOWLEDGMENTS

We thank Hao-Chieh Chiu and Yung-Chi Chang (National Taiwan University) for sharing C. indologenes and P. aeruginosa and the Imaging Core, First Core Laboratory, National Taiwan University College of Medicine for expert assistance on microscopy. Some of the strains used in this study are provided by the Caenorhabditis Genetics Center, which is supported by the NIH Office of Research Infrastructure Programs (P40OD010440), and by the National BioResources Project (NBRP), which is supported by the Japanese Government. Gene database and bioinformatics analysis are provided by WormBase, which is supported by grant #U24 HG002223 from the National Human Genome Research Institute at the US National Institutes of Health, by the UK Medical Research Council, and by the UK Biotechnology and Biological Sciences Research Council. This work was supported by the Taiwan National Science and Technology Council and Taiwan Ministry of Education Featured Areas Research Center Program within the framework of the Higher Education Sprout Project to C.-L.P. (NSTC 110-2634-F-002-017, NSTC 110-2320-B-002-055-MY3, NSTC 112-2320-B-002-018-MY3, and MOE 112L90 1402A); The Baillet Latour Fund, the European Research Council, and the Research Foundation, Flanders to I.B. (ERC grant 950328 and FWO grant G036524N); and a US National Institutes of Health grant and US National Science Foundation award (R35GM131877 and 2032101) to F.C.S.

Footnotes

DECLARATION OF INTERESTS

The authors declare no competing interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS2130171-supplement-1.pdf^{(7.5MB, pdf)}

Data Availability Statement

All the primary data for this study have been deposited at Figshare and are publicly available as of the date of publication, with the DOI in the key resources table.
This study does not use original code for data analysis.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

KEY RESOURCES TABLE.

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
GFP Antibody (B-2)	Santa Cruz Biotechnology	sc-9996
beta actin Antibody (C4)	Santa Cruz Biotechnology	sc-47778
beta tubulin Antibody (2G7D4)	GenScript	A01717
HRP Goat anti-mouse IgG Antibody	BioLegend	405306
GFP-Trap^® Agarose	ChromoTek	AB_2631357
Bacterial and virus strains
E. coli: Strain OP50	Caenorhabditis Genetics Center (CGC)	WormBase: OP50
E. coli: Strain HT115	Caenorhabditis Genetics Center (CGC)	N/A
P. aeruginosa: Strain PA14		N/A
Chryseobacterium indologenes	ATCC	29897
Chemicals, peptides, and recombinant proteins
Antimycin A	Sigma-Aldrich	A8674
IPTG (isopropyl b-D-1-thiogalactopyranoside)	Sigma-Aldrich	I6758
GENEzol	Geneaid	GZX100/D100
Sphingosine-1-phosphate (d18:1)	Cayman Chemical	62570
Benzaldehyde	Sigma-Aldrich	B1334
1-Octanol	Alfa Aesar	A15977
2-Nonanone	Sigma-Aldrich	108731
C16 Ceramide (d18:1/16:0)	Cayman Chemical	10681
S1P-biotin	Echelon Bioscience	S-200B
Streptavidin-HRP conjugate	Invitrogen^™	S911
Deposited data
Raw data of behavior assays	This paper	https://doi.org/10.6084/m9.figshare.24316951.v1
Experimental models: Cell lines
Hamster: CHO cells	ATCC	CCL-61
Xenopus laevis oocytes	Ecocyte Bio Science	N/A
Experimental models: Organisms/strains
C. elegans: Strain N2: wild isolate	CGC	WormBase: N2
C. elegans: Strain VC916: sphk-1(ok1097)/II	CGC	WormBase: sphk-1
C. elegans: Strain RB1465: C23H3.4(ok1693)/II	CGC	WormBase: sptl-1
C. elegans: Strain VC2358: sptl-2(ok2753)/V	CGC	WormBase: sptl-2
C. elegans: Strain VC747: lagr-1(gk327)/I	CGC	WormBase: lagr-1
C. elegans: Strain RB1036: hyl-1(ok976)/IV	CGC	WormBase: hyl-1
C. elegans: Strain JCM1: hyl-2(gnv-2)/X	CGC	WormBase: hyl-2
C. elegans: Strain RB1498: hyl-2(ok1766)/X	CGC	WormBase: hyl-2
C. elegans: Strain VC242: spl-2(ok490)/V	CGC	WormBase: spl-2
C. elegans: Strain CLP1511: sphk-1(twn21) linker gfp/II; twnEx704(Pelt-2::tomm20::mCherry, Pgcy-8::mCherry)	This Paper	N/A
C. elegans: Strain CLP1213: sphk-1(ok1097)/II; twnEx525(Pdpy-7::sphk-1, Pdpy-7::gfp)	This Paper	N/A
C. elegans: Strain CLP1214: sphk-1(ok1097)/II; twnEx526(Pelt-2::sphk-1, Pelt-2::gfp)	This Paper	N/A
C. elegans: Strain CLP1395: sphk-1(ok1097)/II; twnEx633(Punc-119::sphk-1, Pgcy-8::gfp)	This Paper	N/A
C. elegans: Strain CLP1494: sphk-1(ok1097)/II; twnEx681(Pelt-2::sphk-1(Δcam), Pelt-2::gfp)	This Paper	N/A
C. elegans: Strain CLP1495: sphk-1(ok1097)/II; twnEx682(Pelt-2::sphk-1(KD), Pelt-2::gfp)	This Paper	N/A
C. elegans: Strain UP3047: csEx603(Plpr-3::ssSfGFP::lpr-3, Plin-48::mRFP)	Meera Sundaram’s Lab	N/A
C. elegans: Strain CLP1178: twnEx509(Pelt-2::Cas9, PU6::lpr-3 sgRNA, Pelt-2::gfp)	This Paper	N/A
C. elegans: Strain CLP1211: twnEx523(Pdpy-7::Cas9, PU6::lpr-3 sgRNA, Pdpy-7::gfp)	This Paper	N/A
C. elegans: Strain CLP1398: sphk-1(1097)/II; twnEx509(Pelt-2::Cas9, PU6::lpr-3 sgRNA, Pelt-2::gfp)	This Paper	N/A
C. elegans: Strain CLP1399: sphk-1(1097)/II; twnEx523(Pdpy-7::Cas9, PU6::lpr-3 sgRNA, Pdpy-7::gfp)	This Paper	N/A
C. elegans: Strain CLP1469: srsx-22(twn20)/V	This Paper	N/A
C. elegans: Strain CLP1294: F10D7.1(twn15)/X	This Paper	N/A
C. elegans: Strain FX04341: F57A8.4(tm4341)/V	NBRP	WormBase: npr-39
C. elegans: Strain CLP1400: sphr-1(twn18)/V	This Paper	N/A
C. elegans: Strain CLP1512: twnEx705(Psphr-1::mCherry, Ptbh-1::Neongreen, Punc-17::bfp)	This Paper	N/A
C. elegans: Strain CLP1426: sphr-1(twn18)/V; twnEx642(Psphr-1::sphr-1, Psphr-1::mCherry)	This Paper	N/A
C. elegans: Strain CLP1427: sphr-1(twn18)/V; twnEx643(Punc-17::sphr-1, Psphr-1::mCherry)	This Paper	N/A
C. elegans: Strain CLP1428: sphr-1(twn18)/V; twnEx644(Ptbh-1::sphr-1, Psphr-1::mCherry, Ptbh-1::t2a::NG)	This Paper	N/A
C. elegans: Strain CLP1418: sphk-1(ok1097)/II; sphr-1(twn18)/V	This Paper	N/A
C. elegans: Strain CLP1474: sphr-1(twn18)/V; twnEx673(Ptbh-1::hS1PR2, Pgcy-8::mCherry)	This Paper	N/A
C. elegans: Strain CLP1475: sphr-1(twn18)/V; twnEx674(Ptbh-1::hS1PR3, Pgcy-8::mCherry)	This Paper	N/A
C. elegans: Strain CLP1656: sphr-1(twn18)/V; twnEx804(Ptbh-1::SPHR-1(4A), Pgcy-8::gfp)	This Paper	N/A
C. elegans: Strain CLP1251: twnEx557(Ptbh-1::mcasp1, Podr-1::gfp)	This Paper	N/A
C. elegans: Strain CLP1396: sphk-1(ok1097)/II; twnEx557(Ptbh-1::mcasp1, Podr-1::gfp)	This Paper	N/A
C. elegans: Strain CLP1425: sphr-1(twn18)/V; twnEx557(Ptbh-1::mcasp1, Podr-1::gfp)	This Paper	N/A
C. elegans: Strain FX17797: ser-6(tm2146)/IV	NBRP	WormBase: ser-6
C. elegans: CLP1577: ser-6(tm2146)/IV; sphr-1(twn18)/V	This Paper	N/A
C. elegans: Strain CLP1335: twnEx608(Ptbh-1::GCaMP6s, Ptdc-1::bfp)	This Paper	N/A
C. elegans: Strain CLP1397: sphk-1(ok1097)/II; twnEx608(Ptbh-1::GCaMP6s, Ptdc-1::bfp)	This Paper	N/A
C. elegans: Strain CLP1429: sphr-1(twn18)/V; twnEx608(Ptbh-1::GCaMP6s, Ptdc-1::bfp)	This Paper	N/A
C. elegans: Strain CLP1501: sphr-1(twn18)/V; twnEx608(Ptbh-1::GCaMP6s, Ptdc-1::bfp), twnEx684(Ptdc-1::sphr-1::SL2::bfp)	This Paper	N/A
C. elegans: Strain CLP1306: twnEx766(Pelt-2::gfp)	This Paper	N/A
C. elegans: Strain FK181: ksIs2(Pdaf-7::GFP, rol-6(su1006))	CGC	WormBase: ksIs2
C. elegans: Strain CLP1554; ksIs2(Pdaf-7::GFP, rol-6(su1006)); twnEx733(Pstr-3::mCherry, Punc-17::bfp)	This Paper	N/A
C. elegans: Strain MT9455: tbh-1(n3247)/X	CGC	WormBase: tbh-1
Oligonucleotides
smFISH probes for lpr-3, see Table S1	LGC Biosearch Technologies	N/A
sgRNA sequence: lpr-3: ACGACGTTAGCTGTGGCACT	This Paper	N/A
sgRNA sequence: sphr-1: TCTCGACAACATGTCATCGG	This Paper	N/A
sgRNA sequence: srsx-22: AGTATCAAACGAAACAATGG	This Paper	N/A
sgRNA sequence: F10D7.1: GTACACATTGACCAGTAACG	This Paper	N/A
crRNA sequence: sphk-1: CTAGGCAGTTGATGAGAAAA	This Paper	N/A
Primers for Recombinant DNA and genotyping, see Table S1	This Paper	N/A
Recombinant DNA
Pelt-2::tomm20::mCherry	This paper	N/A
Pgcy-8::mCherry	This paper	N/A
Pdpy-7::sphk-1	This paper	N/A
Pdpy-7::gfp	This paper	N/A
Pelt-2::sphk-1	This paper	N/A
Pelt-2::gfp	This paper	N/A
Punc-119::sphk-1	This paper	N/A
Pgcy-8::gfp	This paper	N/A
Pelt-2::sphk-1(Δcam)	This paper	N/A
Pelt-2::sphk-1(KD)	This paper	N/A
Pelt-2::Cas9	This paper	N/A
PU6::lpr-3 sgRNA	This paper	N/A
Pdpy-7::Cas9	This paper	N/A
Psphr-1::mCherry	This paper	N/A
Ptbh-1::Neongreen	This paper	N/A
Punc-17::bfp	This paper	N/A
Psphr-1::sphr-1	This paper	N/A
Punc-17::sphr-1	This paper	N/A
Ptbh-1::sphr-1	This paper	N/A
Ptbh-1::hS1PR2	This paper	N/A
Ptbh-1::hS1PR3	This paper	N/A
Ptbh-1::sphr-1(alkyl mutant)	This paper	N/A
Ptbh-1::mcasp1	This paper	N/A
Podr-1::gfp	This paper	N/A
Ptbh-1::GCaMP6s	This paper	N/A
Ptdc-1::bfp	This paper	N/A
Ptdc-1::sphr-1::SL2::bfp	This paper	N/A
Pstr-3::mCherry	This paper	N/A
Software and algorithms
ImageJ	NIH	https://imagej.nih.gov/ij/
WormLab	MBF Bioscience	https://www.mbfbioscience.com/
The PyMOL Molecular Graphics System	Schriödinger, LLC	https://pymol.org/2/
Metaboseek	Cornell University	https://metaboseek.com/
Xcalibur QualBrowser v4.1.31.9	Thermo Fisher Scientific	https://www.thermofisher.com/
SPARKCONTROL	TECAN	https://www.tecan.com/
Roboocyte2+	Multi Channel Systems MCS GmbH	https://www.multichannelsystems.com/
Prism 10	Graphpad	https://www.graphpad.com/
AlphaFold2	Google DeepMind	https://deepmind.google/technologies/alphafold/
ZEN Microscopy Software	Zeiss	https://www.zeiss.com/microscopy/en/home.html
Metamorph	Molecular Devices	https://www.moleculardevices.com

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PERMALINK

Intercellular sphingolipid signaling mediates aversive learning in C. elegans

Yu-Chun Wu

Isabel Beets

Bennett William Fox

Diana Fajardo Palomino

Li Chen

Chien-Po Liao

Elke Vandewyer

Liang-Yi Lin

Chun-Wei He

Li-Tzu Chen

Chih-Ta Lin

Frank C Schroeder

Chun-Liang Pan

SUMMARY

In brief

Graphical Abstract

INTRODUCTION

RESULTS

SPHK-1/sphingosine kinase regulates stress-induced aversive learning

Figure 1. S1P signaling is required for stress-induced aversive learning.

S1P acts in aversive learning under stress

Figure 2. S1P signaling acts in the acquisition of aversive memory.

Mitochondrial localization of SPHK-1 is required for aversive learning

Lipocalin LPR-3 is an S1P chaperone in aversive learning

Figure 3. The lipocalin LPR-3 is an S1P chaperone.

The C. elegans C24B5.1/sphr-1 encodes an S1PR

Figure 4. The GPCR SPHR-1 acts in RIC neurons to regulate aversive learning.

Sphingolipid signaling remodels RIC properties under mitochondrial stress

Figure 5. S1P promotes stress-induced RIC responses through SPHR-1.

Sphingolipid signaling promotes avoidance of the pathogen C. indologenes

Figure 6. Gut/hypodermal-derived S1P promotes learned aversion of Chryseobacterium indologenes.

DISCUSSION

Figure 7. Schematic model of intercellular sphingolipid signaling.

Sphingolipid signaling encodes an internal state of mitochondrial stress

Sphingolipid signaling promotes learned pathogen avoidance

RESOURCE AVAILABILITY

Lead contact

Materials availability

Data and code availability

KEY RESOURCES TABLE.

STAR★METHODS

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Animals

Microbe Strains

Cell Lines

METHOD DETAILS

Molecular Biology and Germline Transformation

CRISPR/Cas9 Genome Editing

Preparation of the dsDNA asymmetric donors

Preparation of the injection mixtures

Bacterial Avoidance Assay with Antimycin A

Avoidance Assay with RNAi

Bacterial Chemotaxis Assay

Odorant Chemotaxis Assay

S1P and Ceramide Supplement

Metabolomic analysis of worms using HPLC-HRMS

Egg Preparation

Worm Culture

Metabolomics Sample Preparation

HPLC Coupled with HRMS

smFISH

S1P Binding Assay

Calcium Mobilization Assay for SPHR-1 Activation by S1P

Receptor Expression in CHO/mtAEQ/Gα16 Cells

TEVC Recording

Epifluorescent and Confocal Microscopy

Calcium Imaging in a Microfluidic Device

Locomotion Analysis

Western Blotting

Modeling of Protein Structures by AlphaFold2

QUANTIFICATION AND STATISTICAL ANALYSIS

Supplementary Material

Highlights.

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

Receptor Expression in CHO/mtAEQ/Gα₁₆ Cells