Communication between the gut and the nervous system coordinate aspects of behavior and response to stressors. When actively feeding, C. elegans executes a digestive motor program triggered by a calcium wave that requires gap junctional connections along the intestinal length. Loss of function of the innexin-16 (inx-16) gap junctional subunit leads to altered group feeding behavior, leading to social feeding characterized by increased body contact, or clumping, between animals. Using genetic analysis, our data shows that inx-16 genetically interacts with npr-1 and flp-21, but not daf-7. Thus, gut to brain signaling influences social versus solitary feeding behavioral choice.
","acknowledgements":"We thank the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), for several strains, and the National BioResource Project (NBRP) which is funded by the Japanese government for the inx-16 (tm1589) mutant, and the Zhao-Wen Wang lab for sharing a strain. Taylor Allen's generous sharing of equipment and Amy Vashlishan Murray's helpful insights and fruitful discussions are also appreciated.
","authors":[{"affiliations":["IQ Solutions, Inc, Rockville, MD, USA "],"departments":["Science Communications"],"credit":["formalAnalysis","investigation","writing_reviewEditing","methodology","conceptualization"],"email":"learmanlisa@gmail.com","firstName":"Lisa ","lastName":"Calabro","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Oberlin College"],"departments":["Biology"],"credit":["conceptualization","formalAnalysis","project","supervision","methodology","visualization","writing_originalDraft"],"email":"mpeters@oberlin.edu","firstName":"Maureen","lastName":"Peters","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by the Oberlin College Biology Department.
","image":{"url":"https://portal.micropublication.org/uploads/9bc095153b03b4e74f292b7df5f6ebeb.jpeg"},"imageCaption":"inx-16 loss of function increase aggregation and genetic interaction assays show an interaction between flp-21 and npr-1 but not daf-7: (A) Aggregation fractions for N2; npr-1(ad690); inx-16 (tm1589) mutant with inx-16(+) expressed in the muscle (mus.) and inx-16 mutant with inx-16(+) expressed in the adult intestinal (int), both labeled as + “rescue”, n=9 for all strains. (B) Aggregation fractions for defecation mutants compared to N2, mutants used: aex-2(sa3); exp-1(sa6); pbo-4(ok583); pbo-5(ox4); n=6 for all strains. (C) Aggregation fractions for single and double mutants of the following genes: daf-7(e1372); inx-16 (tm1589); npr-1(ad609) alongside N2; n=6 for all strains. (D) Aggregation fractions for single and double mutants of the following genes: flp-21(ok889); inx-16 (tm1589) alongside N2; n=8 for all strains. Statistical significance: *p<0.05; **= p<0.01, ***<0.001; ****<0.0001, ns is not significant.
","imageTitle":"inx-16 mutation results in enhanced aggregation and genetically interacts with both nlp-1 and flp-21
","methods":"Strain maintenance and construction
C. elegans strains were cultured on standard NGM plates seeded with OP50 bacteria using standard protocols. Double daf-2 and npr-1 mutant strains were generated using standard genetic crossing, selection based on dauer or aggregation phenotype, followed by sequencing to confirm genotypes. For flp-21, the large deletion allowed the use of PCR tracking prior to sequencing.
Social feeding assay
Young adult animals were assayed blindly using a protocol adapted from De Bono and Bargmann 1998 (de Bono and Bargmann, 1998). NGM plates were seeded with 200 mL of OP50 in a circular lawn about 25 mm in diameter. Approximately 150 well-fed day 1 adult worms were added and left at 20-22 o C for 2 hours. Then, the fraction of animals in contact with two or more other animals along at least 50% of their body length was tallied by visual inspection. For each panel, the assays were completed in a short time period for all genotypes shown.
Statistical analysis and graphing
For statistical analysis, Graphpad/Prism (Version 11 for macOS) was used. Each panel's data set was tested for Gaussian distribution. Only one panel's data set (A) showed some non-Gaussian distribution, so Kruskal-Wallis program was used for panel A's ANNOVA, and multiple comparisons tests. For the remaining panels, one-way ANNOVA analysis was followed by multiple comparisons using Tukey's test. Statistical deviation was considered significant if p<0.05. Bar graphs show all data points, mean and standard error of the mean. p value scores are listed for informative comparisons relevant to our study. Differences between npr-1 and N2, or other mutants that were previously published and/or strikingly obvious were not specifically noted. For example, statistical tests between N2 and npr-1, inx-16 and npr-1, npr-1 and daf-7 were not highlighted to simplify the figures.
","reagents":"Strain | Genotype | Available from |
Bristol isolate | CGC | |
National BioResource Project (NBRP) | ||
| ||
CGC | ||
CGC | ||
CGC | ||
CGC | ||
CGC | ||
CGC | ||
CGC | ||
| ||
| ||
inx-16(tm1589)I; flp-21(ok889) V
|
| |
| Zhao-Wen Wang Lab |
Organisms adapt to their dynamic internal and external environments by leveraging systemic physiological responses using signaling pathways involving multiple tissues and extensive cross-talk. Recently, the gut has emerged as an important signaling center keenly attuned to animal's feeding state, food quality, and pathogenic exposure. Within C. elegans, gut-derived secreted peptide signals influence feeding, stress response, waste release and avoidance behavior by acting on neurons of the enteric and central nervous systems (Artan et al., 2016; Jia et al., 2024; Sural et al., 2025). For example, INS-7 and INS-35 regulate the NSM neuron to adjust pharyngeal pumping rates (Sural et al., 2025). Additional insulin family members such as INS-3 and INS-11 function in gut to brain signaling pathways (Lee and Mylonakis, 2017; Veuthey et al., 2025). Several non-insulin peptides are also involved. FLP-2 (FMRFamide-like peptide) acts via the interneuron AIY to induce responses to oxidative stress within the animal (Jia et al., 2024). NLP-40 (Neuropeptide-like protein) is released to instruct the posterior enteric neurons, AVL and DVB, to elicit steps of the digestive/defecation motor program; its release is tied to a periodic calcium wave that occurs in well-fed animals with a regular frequency of 45-55 seconds (Choi et al., 2023; Mahoney et al., 2008; Wang et al., 2013).
This regular calcium wave may trigger release of additional peptides that influence nervous system behavior more broadly. Several mutants with poor digestive waste release originally identified due to their constipated phenotypes turn out to encode integral components of peptide processing and release such as egl-3, egl-21, aex-4, and aex-5 (Husson et al., 2007; Khawand et al., 2026; Mahoney et al., 2008). At least one peptide, NLP-40, is released in time with the digestive motor program, via activation of synaptotagmin-2 (snt-2), a calcium-sensing protein that triggers DCV fusion (Wang et al., 2013). The tyramine response pathway offers additional evidence of calcium-triggered peptide release (Veuthey et al., 2025). This enticing evidence suggests that the rhythmic calcium flux that triggers NLP-40 release, and other steps of the digestive/defecation motor program, may be used more broadly to influence behavioral choices where feeding and nutritional status is relevant. With this notion in mind, we explored an observation of social feeding behavior in a mutant that alters intestinal calcium wave dynamics.
The innexin-16 (inx-16) gap junction subunit is critical for period intestinal calcium waves associated with the digestive motor program; loss of function inx-16 mutants disrupt the intestinal calcium wave and greatly reduce the digestive motor program steps that require NLP-40 release (Peters et al., 2007). During routine maintenance of inx-16(tm1587), we observed increased aggregation of well-fed worms and became intrigued. To explore this further, we assayed for social feeding behavior quantitatively. For comparison we included N2, a solitary strain, and a social feeding strain, npr-1(ad609) The gene, npr-1, encodes a C. elegans receptor related to neuropeptide Y, and is neuronally expressed. When active, NPR-1 inhibits the tendency of animals to aggregate in groups, suppresses oxygen level preferences and affects bordering (de Bono and Bargmann, 1998; Gray et al., 2004). The npr-1 allele we used, ad609, has been characterized to be a strong loss of function allele, equivalent to a genetic null (de Bono and Bargmann, 1998). Our results show that inx-16 mutant animals maintain contact with other animals more frequently than wild-type (N2) solitary worms; young adult inx-16(tm1589) animals demonstrated a significant difference in the number of inx-16 animals found in direct contact with their peers compared to N2 (~0% vs. ~15%; Figure 1A). Yet, the inx-16 phenotype is much weaker than alleles of npr-1 (ad609) allele (~80% vs. ~15%; Figure 1A).
To verify that INX-16 functions within the intestine to cause the observed tendency to aggregate, we employed tissue specific rescue of inx-16 mutants. Our and others' inx-16 expression analysis indicated that inx-16 was only expressed in the intestine, yet two studies have provided phenotypic evidence suggesting additional areas of function (Bhattacharya et al., 2019; Liu et al., 2013; Sojka et al., 2025). Intestine-specific rescue was achieved using a construct with a short, well-defined fragment, the sequence of the egg yolk protein, vitellogenin-2, (p-Vit-2) gene, placed upstream of an INX-16 translational fusion (MacMorris et al., 1992). Prior work using this construct led to full rescue of the defecation defects in adults (Peters et al., 2007). Likewise, solitary feeding behavior was restored in the inx-16+pVit-2 intestinal rescue line (Figure 1A). By contrast, driving expression of inx-16 in body wall muscles, using the myo-3 promoter, was unable to restore solitary (wild-type) feeding behavior (Liu et al., 2013). These data suggest that intestinal INX-16 function contributes to the animal's feeding behaviors.
Having shown that inx-16's actions in the intestine can rescue the social feeding phenotype, we queried whether aggregated feeding was a common feature of mutants with impaired digestive waste release (Thomas, 1990). Defecation mutants induce constipation. One constipated mutant with defects in the expulsion step, exp-1, disrupts an excitatory GABA-ergic receptor in enteric muscles and has been shown to mildly increase social feeding behavior (Beg and Jorgensen, 2003; Bendesky et al., 2012). We assayed this mutant, and others, selecting set of mutants that affect each defecation motor step and all tissue types involved in the motor program (Branicky and Hekimi, 2006; Thomas, 1990). Two genes required for the first motor step (the posterior body contraction) the intestinal sodium proton exchanger, pbo-4/nhx-7, and the downstream proton-gated ion channel, pbo-5, localized to body wall muscles, were tested (Beg et al., 2008; Pfeiffer et al., 2008). Neither of these Pbo mutants showed increases in social feeding in comparison to N2 (Figure 1B). The later steps of the program, anterior body contraction and enteric muscle contraction, require NLP-40 release from the intestine to stimulate GABA release of from enteric neurons, AVL and DVB (Beg et al., 2008; Wang et al., 2013). NLP-40 binding activates AEX-2, a g-protein coupled receptor, on these neurons (Allman et al., 2016; Mahoney et al., 2008). Like the Pbo mutants, aex-2(sa3) showed no significant increase over wild-type in our aggregation assay (Figure 1B). In our assays, exp-1(sa6) did not increase aggregation which differs from prior findings that reported a small but significant increase (Bendesky et al., 2012). Though our defecation mutant panel is not exhaustive, these data suggest that the social feeding phenotype of inx-16 is not broadly shared with mutants suffering from constipation; disrupting defecation per se does not alter social feeding behavior tendencies in C. elegans.
Two key gene pathways control the choice between solitary and social feeding behavior: npr-1 and daf-7. Therefore, we used genetic assays to elucidate potential connections between inx-16 and these known pathways (de Bono and Bargmann, 1998; de Bono et al., 2002). Double mutants of inx-16 with daf-7(e1372) III and npr-1(ad690) X were constructed and tested alongside single mutants to determine whether the double mutant lines had enhanced aggregation. The combination of inx-16 I and daf-7(e1372) III enhanced aggregation substantially, with the inx-16;daf-7 double mutant exhibiting increased aggregation compared to the daf-7 mutant alone, approximately 23% for the double compared to ~13% for either single mutants (Figure 1C). This additive effect is consistent with inx-16 functioning at least partly independently of daf-7. By contrast the combination of inx-16 and the strong loss of function npr-1 allele, ad690, did not augment the social feeding level relative to that of npr-1 alone. The inx-16;npr-1 double mutants exhibit no increase in aggregation compared to the npr-1 mutant alone (60% for the double compared to 58% for the single mutant, Figure 1C). These findings are consistent with inx-16 functioning via the npr-1 pathway to mediate social feeding behavior.
Since our findings place inx-16 within the npr-1 pathway rather than the daf-7 pathway, we searched for NPR-1 ligands with evidence of intestinal expression. Of the two identified NPR-1 ligands, FLP-18 and FLP-21, flp-21 showed intestinal expression in addition to a subset of neurons (Kubiak et al., 2003; Rogers et al., 2003). The flp-21 mutation causes a significantly increased propensity for animals to aggregate, enhancing aggregation to ~15% (Figure 1D). Interestingly, flp-21's magnitude of impact on feeding behavior is similar to that of inx-16 mutation, ~15% for flp-21 vs. ~14% for inx-16 (Figure 1D, (Rogers et al., 2003)). Thus, we hypothesized that FLP-21 could be released from the intestine following calcium waves in a manner analogous to NLP-40 release. To begin to investigate this model, we investigated epistasis between inx-16 and flp-21. The double mutant line exhibited an aggregation profile very similar to that of either individual mutant strain (inx-16;flp-2, ~ 13%; Figure 1D). Our finding is consistent with a model placing FLP-21 release downstream of inx-16 function, and by inference the rhythmic intestinal calcium wave that directs the digestive motor program (Dal Santo et al., 1999; Espelt et al., 2005; Teramoto and Iwasaki, 2006) .
Due to flp-21's reported intestinal localization, we suggest that inx-16 social feeding phenotype may be due to reduced FLP-21 release from the intestine. Since NPR-1 signaling dampens social feeding, loss of FLP-21 would increase aggregation, as seen in both inx-16 and flp-21 mutants. Our model is supported by the previously reported epistasis analysis of flp-21(pk1601) and npr-1 (ad690); this flp-21 allele did not enhance npr-1(ad690)'s social feeding level, matching our results (Figure 1C; (Rogers et al., 2003)). Given that flp-18, the other known NPR-1 ligand, is not expressed in the intestine, flp-18 presumably remains active in the inx-16 mutant, allowing ample residual suppression of the social feeding trait, resulting in the modest aggregation seen in both the inx-16 and flp-21 mutants (Figure 1D; (Kapahi et al., 2010)).
In summary, our findings demonstrate a behavioral feeding phenotype associated with disrupted intestinal calcium wave dynamics. We propose that this phenotype is due to disruption of FLP-21 mediated activation of NPR-1. Moreover, the social feeding phenotype of inx-16 suggests that the periodic intestinal calcium waves serve to regulate peptide release broadly, influencing a host of behaviors beyond defecation and adding to the growing body of evidence illustrating the importance of gut to brain signaling for animal behavior.
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","pubmedId":"","doi":"10.1016/j.cub.2013.03.049"}],"title":"Gap junctional communication in the intestine affects feeding behavior via FLP-21 and the NPR-1 mediated signaling pathway
","reviews":[{"reviewer":{"displayName":"Surojit Sural"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":null}]},{"id":"9f4c2932-14a4-46a3-a044-2082f7dbdb84","decision":"accept","abstract":"Communication between the gut and the nervous system coordinates aspects of behavior and responses to stressors. When actively feeding, C. elegans executes a digestive motor program triggered by a calcium wave that requires gap junctional connections along the intestinal length. Loss of function of the innexin-16 (inx-16) gap junctional subunit results in altered group feeding behavior, leading to social feeding characterized by increased body contact, or clumping, between animals. Using genetic analysis, our data shows that inx-16 genetically interacts with npr-1 and flp-21, but not daf-7. Thus, gut-to-brain signaling influences social versus solitary feeding behavioral choice.
","acknowledgements":"We thank the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), for several strains, and the National BioResource Project (NBRP) which is funded by the Japanese government for the inx-16 (tm1589) mutant, and the Zhao-Wen Wang lab for sharing a strain. Taylor Allen's generous sharing of equipment and Amy Vashlishan Murray's helpful insights and fruitful discussions are also appreciated.
","authors":[{"affiliations":["IQ Solutions, Inc, Rockville, MD, USA "],"departments":["Science Communications"],"credit":["formalAnalysis","investigation","writing_reviewEditing","methodology","conceptualization"],"email":"learmanlisa@gmail.com","firstName":"Lisa ","lastName":"Calabro","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Oberlin College"],"departments":["Biology"],"credit":["conceptualization","formalAnalysis","project","supervision","methodology","visualization","writing_originalDraft"],"email":"mpeters@oberlin.edu","firstName":"Maureen","lastName":"Peters","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0001-0337-0790"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by the Oberlin College Biology Department.
","image":{"url":"https://portal.micropublication.org/uploads/05794fc712c685f84ac659d5bd0dca96.jpeg"},"imageCaption":"Figure 1: inx-16 loss of function increases aggregation, and genetic interaction assays show an interaction between flp-21 and npr-1 but not daf-7. (A) Aggregation fractions for N2; npr-1(ad690); inx-16(tm1589) mutant with inx-16 (+) expressed in the muscle (mus.) and inx-16 mutant with inx-16 (+) expressed in the adult intestinal (int), both labeled as + “rescue”, n=9 for all strains. (B) Aggregation fractions for defecation mutants compared to N2, mutants used: aex-2(sa3); exp-1(sa6); pbo-4(ok583); pbo-5(ox4); n=6 for all strains. (C) Aggregation fractions for single and double mutants of the following genes: inx-16(tm1589); npr-1(ad609); daf-7(e1372); n=6 for all strains. (D) Aggregation fractions for single and double mutants of the following genes: flp-21(ok889); inx-16(tm1589) alongside N2; n=8 for all strains. Statistical significance: *p<0.05; **= p<0.01; ***<0.001; ****<0.0001; ns is not significant. Brackets used for comparisons between two mutants; line used across three strains for genetic interactions.
","imageTitle":"inx-16 mutation results in enhanced aggregation and genetically interacts with both nlp-1 and flp-21
","methods":"Strain maintenance and construction
C. elegans strains were cultured on standard NGM plates seeded with OP50 bacteria using standard protocols. Double daf-2 and npr-1 mutant strains were generated using standard genetic crossing, selection based on dauer or aggregation phenotype, followed by sequencing to confirm genotypes. For flp-21, the large deletion allowed the use of PCR tracking prior to sequencing.
Social feeding assay
Young adult animals were assayed blindly using a protocol adapted from De Bono and Bargmann (1998). Strains containing the daf-2(e1372) mutation were reared at 15o C to prevent dauer formation. NGM plates were seeded with 200 mL of OP50 in a circular lawn about 25 mm in diameter. Approximately 150 well-fed day 1 adult worms were added and left at 20-22o C for 2 hours. Then, the fraction of animals in contact with two or more other animals along at least 50% of their body length was tallied by visual inspection. For each panel's experiments, the full set of strains was tested each assay day and the full set of assays were completed in a short time period.
Statistical analysis and graphing
For statistical analysis, Graphpad/Prism (Version 11 for macOS) was used. Each panel's data set was tested for Gaussian distribution. Only one panel's data set (A) showed some non-Gaussian distribution, so Kruskal-Wallis program was applied. Otherwise, ANOVA analysis was followed by multiple comparisons using Tukey's test (one-way ANOVA for panels A and B; two-way ANOVA for panels C and D). Statistical deviation was considered significant if p<0.05. Bar graphs show all data points, mean and standard error of the mean. p value scores are listed for informative comparisons relevant to our study. Differences between npr-1 and N2, or other mutants that were previously published and/or strikingly obvious were not specifically noted. For example, statistical tests between N2 and npr-1, inx-16 and npr-1, npr-1 and daf-7 were not highlighted to simplify the figures.
","reagents":"Strain | Genotype | Available from |
Bristol isolate | CGC | |
National BioResource Project (NBRP) | ||
| ||
CGC | ||
CGC | ||
CGC | ||
CGC | ||
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inx-16(tm1589) I; zwEx701[Pmyo-3::inx-16(+)::GFP + Pmyo-3::GFP] | Zhao-Wen Wang Lab |
Organisms adapt to their dynamic internal and external environments by leveraging systemic physiological responses using signaling pathways involving multiple tissues and extensive cross-talk. Recently, the gut has emerged as an important signaling center keenly attuned to animal's feeding state, food quality, and pathogenic exposure. Within C. elegans, gut-derived secreted peptide signals influence feeding, stress response, waste release and avoidance behavior by acting on neurons of the enteric and central nervous systems (Artan et al., 2016; Jia et al., 2024; Sural et al., 2025). For example, INS-7 and INS-35 regulate the NSM neuron to adjust pharyngeal pumping rates (Sural et al., 2025). Additional insulin family members such as INS-3 and INS-11 function in gut-to-brain signaling pathways (Lee and Mylonakis, 2017; Veuthey et al., 2025). Several non-insulin peptides are also involved. FLP-2 (FMRFamide-like peptide) acts via the interneuron AIY to induce responses to oxidative stress within the animal (Jia et al., 2024). NLP-40 (Neuropeptide-like protein) is released to instruct the posterior enteric neurons, AVL and DVB, to elicit steps of the digestive/defecation motor program; its release is tied to a periodic calcium wave that occurs in well-fed animals with a regular frequency of 45-55 seconds (Choi et al., 2023; Mahoney et al., 2008; Wang et al., 2013).
This regular calcium wave may trigger release of additional peptides that influence nervous system behavior more broadly. Several mutants with poor digestive waste release originally identified due to their constipated phenotypes turn out to encode integral components of peptide processing and release such as egl-3, egl-21, aex-1, aex-4, and aex-5 (Husson et al., 2007; Khawand et al., 2026; Mahoney et al., 2008). At least one peptide, NLP-40, is released in time with the digestive motor program, via activation of synaptotagmin-2 (snt-2), a calcium-sensing protein that triggers DCV fusion (Wang et al., 2013). The tyramine response pathway offers additional evidence of calcium-triggered peptide release (Veuthey et al., 2025). This enticing evidence suggests that the rhythmic calcium flux that triggers NLP-40 release, and other steps of the digestive/defecation motor program, may be used more broadly to influence behavioral choices where feeding and nutritional status is relevant. With this notion in mind, we explored an observation of social feeding behavior in a mutant that alters intestinal calcium wave dynamics.
The innexin-16 (inx-16) gap junction subunit is critical for periodic intestinal calcium waves associated with the digestive motor program; loss of function inx-16 mutants disrupt the intestinal calcium wave and greatly reduce the digestive motor program steps that require NLP-40 release (Peters et al., 2007). During routine maintenance of inx-16(tm1587), we observed increased aggregation of well-fed worms and became intrigued. To explore this further, we assayed for social feeding behavior quantitatively. For comparison, we included N2, a solitary strain, and a social feeding strain, npr-1(ad609) The gene, npr-1, encodes a C. elegans receptor related to neuropeptide Y and is neuronally expressed. When active, NPR-1 inhibits the tendency of animals to aggregate in groups, suppresses oxygen level preferences, and affects bordering (de Bono and Bargmann, 1998; Gray et al., 2004). The npr-1 allele we used, ad609, has been characterized to be a strong loss of function allele, equivalent to a genetic null (de Bono and Bargmann, 1998). Our results show that inx-16 mutant animals maintain contact with other animals more frequently than wild-type (N2) solitary worms; young adult inx-16(tm1589) animals demonstrated a significant difference in the number of inx-16 animals found in direct contact with their peers compared to N2 (~0% vs. ~15%; Figure 1A). Yet, the inx-16 phenotype is much weaker than alleles of npr-1(ad609) allele (~80% vs. ~15%; Figure 1A).
To verify that INX-16 functions within the intestine to cause the observed tendency to aggregate, we employed tissue specific rescue of inx-16 mutants. Our and others' inx-16 expression analysis indicated that inx-16 was only expressed in the intestine, yet two studies have provided phenotypic evidence suggesting additional areas of function (Bhattacharya et al., 2019; Liu et al., 2013; Sojka et al., 2025). Intestine-specific rescue was achieved using a construct with a short, well-defined fragment, regulatory sequence of the egg yolk protein, vitellogenin-2, (p-Vit-2) gene, placed upstream of an INX-16 translational fusion (MacMorris et al., 1992). Prior work using this construct led to full rescue of the defecation defects in adults (Peters et al., 2007). Likewise, solitary feeding behavior was restored in the inx-16+pVit-2 intestinal rescue line (Figure 1A). By contrast, driving expression of inx-16 in body wall muscles, using the myo-3 promoter, was unable to restore solitary (wild-type) feeding behavior (Liu et al., 2013). These data suggest that intestinal INX-16 function contributes to the animal's feeding behaviors.
Having shown that inx-16's actions in the intestine can rescue the social feeding phenotype, we queried whether aggregated feeding was a common feature of mutants with impaired digestive waste release (Thomas, 1990). Defecation mutants induce constipation. One constipated mutant with defects in the expulsion step, exp-1, disrupts an excitatory GABA-ergic receptor in enteric muscles and has been shown to mildly increase social feeding behavior (Beg and Jorgensen, 2003; Bendesky et al., 2012). We assayed this mutant, and others, selecting a set of mutants that affect each defecation motor step and all tissue types involved in the motor program (Branicky and Hekimi, 2006; Thomas, 1990). Two genes required for the first motor step (the posterior body contraction), the intestinal sodium proton exchanger, pbo-4/nhx-7, and the downstream proton-gated ion channel, pbo-5, localized to body wall muscles, were tested (Beg et al., 2008; Pfeiffer et al., 2008). Neither of these Pbo mutants showed increases in social feeding in comparison to N2 (Figure 1B). The later steps of the program, anterior body contraction and enteric muscle contraction, require NLP-40 release from the intestine to stimulate GABA release from enteric neurons, AVL and DVB (Wang et al., 2013). NLP-40 binding activates AEX-2, a g-protein coupled receptor, on these neurons (Allman et al., 2016; Mahoney et al., 2008). Like the Pbo mutants, aex-2(sa3) showed no significant increase over wild-type in our aggregation assay (Figure 1B). In our assays, exp-1(sa6) did not increase aggregation, which differs from prior findings that reported a small but significant increase (Bendesky et al., 2012). Though our defecation mutant panel is not exhaustive, these data suggest that the social feeding phenotype of inx-16 is not broadly shared with mutants suffering from constipation; disrupting defecation per se does not alter social feeding behavior tendencies in C. elegans.
Two key gene pathways control the choice between solitary and social feeding behavior: npr-1 and daf-7. Therefore, we used genetic assays to elucidate potential connections between inx-16 and these known pathways (de Bono and Bargmann, 1998; de Bono et al., 2002). Double mutants of inx-16 with daf-7(e1372) and npr-1(ad690) were constructed and tested alongside single mutants to determine whether the double mutant lines had enhanced aggregation. The combination of inx-16 and daf-7(e1372) enhanced aggregation substantially, with the inx-16;daf-7 double mutant exhibiting increased aggregation compared to the daf-7 mutant alone, approximately 23% for the double compared to ~13% for either single mutants (Figure 1C). This additive effect is consistent with inx-16 functioning at least partly independently of daf-7. By contrast, the combination of inx-16 and the strong loss of function npr-1 allele, ad690, did not augment the social feeding level relative to that of npr-1 alone. The inx-16;npr-1 double mutants exhibit no increase in aggregation compared to the npr-1 mutant alone (60% for the double compared to 58% for the single mutant, Figure 1C). The results of a two-way ANOVA analysis support a highly significant genetic interaction between inx-16 and npr-1 (P < 0.003; F = (1, 20)). These findings are consistent with inx-16 functioning via the npr-1 pathway to mediate social feeding behavior.
Since our findings place inx-16 within the npr-1 pathway rather than the daf-7 pathway, we searched for NPR-1 ligands with evidence of intestinal expression. Of the two identified NPR-1 ligands, FLP-18 and FLP-21, flp-21 showed intestinal expression in addition to a subset of neurons (Kubiak et al., 2003; Rogers et al., 2003). The flp-21 mutation causes a significantly increased propensity for animals to aggregate, enhancing aggregation to ~15% (Figure 1D). Interestingly, flp-21's magnitude of impact on feeding behavior is similar to that of inx-16 mutation, ~15% for flp-21 vs. ~14% for inx-16 (Figure 1D; Rogers et al., 2003). Thus, we hypothesized that FLP-21 could be released from the intestine following calcium waves in a manner analogous to NLP-40 release. To begin to investigate this model, we explored epistasis between inx-16 and flp-21. The double mutant line exhibited an aggregation profile very similar to that of either individual mutant strain (inx-16;flp-21, ~ 13%; Figure 1D). Statistical analysis for gene interaction upholds this epistatic relationship (P < 0.0001; F = (1, 28) = 89). Our results support a model placing FLP-21 release downstream of INX-16 function, and by inference the rhythmic intestinal calcium wave that directs the digestive motor program (Dal Santo et al., 1999; Espelt et al., 2005; Teramoto and Iwasaki, 2006).
Due to flp-21's reported intestinal localization, we suggest that inx-16's social feeding phenotype may be due to reduced FLP-21 release from the intestine. Since NPR-1 signaling dampens social feeding, loss of FLP-21 would increase aggregation, as seen in both inx-16 and flp-21 mutants. Our model is supported by the previously reported epistasis analysis of flp-21(pk1601) and npr-1(ad690); this flp-21 allele did not enhance npr-1(ad690)'s social feeding level, matching our results (Figure 1C; Rogers et al., 2003). Given that flp-18, the other known NPR-1 ligand, is not expressed in the intestine, flp-18 presumably remains active in the inx-16 mutant, allowing ample residual suppression of social feeding to result in the modest aggregation seen in both the inx-16 and flp-21 mutants (Figure 1D; Kapahi et al., 2010).
In summary, our findings demonstrate a behavioral feeding phenotype associated with disrupted intestinal calcium wave dynamics. We propose that this phenotype is due to disruption of FLP-21 mediated activation of NPR-1. Moreover, the social feeding phenotype of inx-16 suggests that the periodic intestinal calcium waves serve to regulate peptide release broadly, influencing a host of behaviors beyond defecation and adding to the growing body of evidence illustrating the importance of gut-to-brain signaling for animal behavior.
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","pubmedId":"","doi":"10.1093/genetics/124.4.855"},{"reference":"Veuthey T, Florman JT, Giunti Sn, Romussi S, De Rosa MaJ, Alkema MJ, Rayes D. 2025. The neurohormone tyramine stimulates the secretion of an insulin-like peptide from the Caenorhabditis elegans intestine to modulate the systemic stress response. PLOS Biology 23: e3002997.
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","pubmedId":"","doi":"10.1016/j.cub.2013.03.049"}],"title":"Gap junctional communication in the intestine affects social feeding behavior via FLP-21 and the NPR-1 mediated signaling pathway
","reviews":[{"reviewer":{"displayName":"Surojit Sural"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":"1779398099521"}]},{"id":"332e08cf-6fe7-4e50-9fce-f162696661e3","decision":"accept","abstract":"Communication between the gut and the nervous system coordinates aspects of behavior and responses to stressors. When actively feeding, C. elegans executes a digestive motor program triggered by a calcium wave that requires gap junctional connections along the intestinal length. Loss of function of the innexin-16 (inx-16) gap junctional subunit results in altered group feeding behavior, leading to social feeding characterized by increased body contact, or clumping, between animals. Using genetic analysis, our data shows that inx-16 genetically interacts with npr-1 and flp-21, but not daf-7. Thus, gut-to-brain signaling influences social versus solitary feeding behavioral choice.
","acknowledgements":"We thank the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), for several strains, and the National BioResource Project (NBRP) which is funded by the Japanese government for the inx-16 (tm1589) mutant, and the Zhao-Wen Wang lab for sharing a strain. Taylor Allen's generous sharing of equipment and Amy Vashlishan Murray's helpful insights and fruitful discussions are also appreciated.
","authors":[{"affiliations":["IQ Solutions, Inc, Rockville, MD, USA "],"departments":["Science Communications"],"credit":["formalAnalysis","investigation","writing_reviewEditing","methodology","conceptualization"],"email":"learmanlisa@gmail.com","firstName":"Lisa ","lastName":"Calabro","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Oberlin College"],"departments":["Biology"],"credit":["conceptualization","formalAnalysis","project","supervision","methodology","visualization","writing_originalDraft"],"email":"mpeters@oberlin.edu","firstName":"Maureen","lastName":"Peters","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0001-0337-0790"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by the Oberlin College Biology Department.
","image":{"url":"https://portal.micropublication.org/uploads/05794fc712c685f84ac659d5bd0dca96.jpeg"},"imageCaption":"Figure 1: inx-16 loss of function increases aggregation, and genetic interaction assays show an interaction between flp-21 and npr-1 but not daf-7. (A) Aggregation fractions for N2; npr-1(ad609); inx-16(tm1589) mutant with inx-16 (+) expressed in the muscle (mus.) and inx-16 mutant with inx-16 (+) expressed in the adult intestinal (int.), both labeled as + “rescue”, n=9 for all strains. (B) Aggregation fractions for defecation mutants compared to N2, mutants used: aex-2(sa3); exp-1(sa6); pbo-4(ok583); pbo-5(ox4); n=6 for all strains. (C) Aggregation fractions for single and double mutants of the following genes: inx-16(tm1589); npr-1(ad609); daf-7(e1372); n=6 for all strains. (D) Aggregation fractions for single and double mutants of the following genes: flp-21(ok889); inx-16(tm1589) alongside N2; n=8 for all strains. Statistical significance: *p<0.05; **= p<0.01; ***<0.001; ****<0.0001; ns is not significant. Brackets used for comparisons between two mutants; line used across three strains for genetic interactions.
","imageTitle":"inx-16 mutation results in enhanced aggregation and genetically interacts with both nlp-1 and flp-21
","methods":"Strain maintenance and construction
C. elegans strains were cultured on standard NGM plates seeded with OP50 bacteria using standard protocols. Double daf-2 and npr-1 mutant strains were generated using standard genetic crossing, selection based on dauer or aggregation phenotype, followed by sequencing to confirm genotypes. For flp-21, the large deletion allowed the use of PCR tracking prior to sequencing.
Social feeding assay
Young adult animals were assayed blindly using a protocol adapted from De Bono and Bargmann (1998). Strains containing the daf-2(e1372) mutation were reared at 15o C to prevent dauer formation. NGM plates were seeded with 200 mL of OP50 in a circular lawn about 25 mm in diameter. Approximately 150 well-fed day 1 adult worms were added and left at 20-22o C for 2 hours. Then, the fraction of animals in contact with two or more other animals along at least 50% of their body length was tallied by visual inspection. For each panel's experiments, the full set of strains was tested each assay day and the full set of assays were completed in a short time period.
Statistical analysis and graphing
For statistical analysis, Graphpad/Prism (Version 11 for macOS) was used. Each panel's data set was tested for Gaussian distribution. Only one panel's data set (A) showed some non-Gaussian distribution, so Kruskal-Wallis program was applied. Otherwise, ANOVA analysis followed by multiple comparisons using Tukey's test (one-way ANOVA for panels A and B; two-way ANOVA for panels C and D) was used. Statistical deviation was considered significant if p<0.05. Bar graphs show all data points, mean and standard error of the mean. p value scores are listed for informative comparisons relevant to our study. Differences between npr-1 and N2, or other mutants that were previously published and/or strikingly obvious were not specifically noted. For example, statistical tests between N2 and npr-1; inx-16 and npr-1; npr-1 and daf-7 were not highlighted to simplify the figures.
","reagents":"Strain | Genotype | Available from |
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inx-16(tm1589) I; zwEx701[Pmyo-3::inx-16(+)::GFP + Pmyo-3::GFP] | Zhao-Wen Wang Lab |
Organisms adapt to their dynamic internal and external environments by leveraging systemic physiological responses using signaling pathways involving multiple tissues and extensive cross-talk. Recently, the gut has emerged as an important signaling center keenly attuned to an animal's feeding state, food quality, and pathogenic exposure. Within C. elegans, gut-derived secreted peptide signals influence feeding, stress response, waste release and avoidance behavior by acting on neurons of the enteric and central nervous systems (Artan et al., 2016; Jia et al., 2024; Sural et al., 2025). For example, INS-7 and INS-35 regulate the NSM neuron to adjust pharyngeal pumping rates (Sural et al., 2025). Additional insulin family members such as INS-3 and INS-11 function in gut-to-brain signaling pathways (Lee and Mylonakis, 2017; Veuthey et al., 2025). Several non-insulin peptides are also involved. FLP-2 (FMRFamide-like peptide) acts via the interneuron AIY to induce responses to oxidative stress within the animal (Jia et al., 2024). NLP-40 (Neuropeptide-like protein) is released to instruct the posterior enteric neurons, AVL and DVB, to elicit steps of the digestive/defecation motor program; its release is tied to a periodic calcium wave that occurs in well-fed animals with a regular frequency of 45-55 seconds (Choi et al., 2023; Mahoney et al., 2008; Wang et al., 2013).
This regular calcium wave may trigger release of additional peptides that influence nervous system behavior more broadly. Several mutants with poor digestive waste release originally identified due to their constipated phenotypes turn out to encode integral components of peptide processing and release such as egl-3, egl-21, aex-1, aex-4, and aex-5 (Husson et al., 2007; Khawand et al., 2026; Mahoney et al., 2008). At least one peptide, NLP-40, is released in time with the digestive motor program, via activation of synaptotagmin-2 (snt-2), a calcium-sensing protein that triggers DCV fusion (Wang et al., 2013). The tyramine response pathway offers additional evidence of calcium-triggered peptide release (Veuthey et al., 2025). This enticing evidence suggests that the rhythmic calcium flux that triggers NLP-40 release, and other steps of the digestive/defecation motor program, may be used more broadly to influence behavioral choices where feeding and nutritional status are relevant. With this notion in mind, we explored an observation of social feeding behavior in a mutant that alters intestinal calcium wave dynamics.
The innexin-16 (inx-16) gap junction subunit is critical for periodic intestinal calcium waves associated with the digestive motor program; loss of function inx-16 mutants disrupt the intestinal calcium wave and greatly reduce the digestive motor program steps that require NLP-40 release (Peters et al., 2007). During routine maintenance of inx-16(tm1589), we observed increased aggregation of well-fed worms and became intrigued. To explore this further, we assayed for social feeding behavior quantitatively. For comparison, we included N2, a solitary strain, and a social feeding strain, npr-1(ad609). The gene, npr-1, encodes a C. elegans receptor related to neuropeptide Y and is neuronally expressed. When active, NPR-1 inhibits the tendency of animals to aggregate in groups, suppresses oxygen level preferences, and affects bordering (de Bono and Bargmann, 1998; Gray et al., 2004). The npr-1 allele we used, ad609, has been characterized to be a strong loss of function allele, equivalent to a genetic null (de Bono and Bargmann, 1998). Our results show that inx-16 mutant animals maintain contact with other animals more frequently than wild-type (N2) solitary worms; young adult inx-16(tm1589) animals demonstrated a significant difference in the number of inx-16 animals found in direct contact with their peers compared to N2 (~0% vs. ~15%; Figure 1A). Yet, the inx-16 phenotype is much weaker than the npr-1(ad609) allele (~80% vs. ~15%; Figure 1A).
To verify that INX-16 functions within the intestine to cause the observed tendency to aggregate, we employed tissue specific rescue of inx-16 mutants. Our and others' inx-16 expression analyses indicated that inx-16 was only expressed in the intestine, yet two studies have provided phenotypic evidence suggesting additional areas of function (Bhattacharya et al., 2019; Liu et al., 2013; Sojka et al., 2025). Intestine-specific rescue was achieved using a construct with a short, well-defined fragment, regulatory sequence of the egg yolk protein, vitellogenin-2, (vit-2) gene, placed upstream of an INX-16 translational fusion (MacMorris et al., 1992). Prior work using this construct led to full rescue of the defecation defects in adults (Peters et al., 2007). Likewise, solitary feeding behavior was restored in the inx-16+pVit-2 intestinal rescue line (Figure 1A). By contrast, driving expression of inx-16 in body wall muscles, using the myo-3 promoter, was unable to restore solitary (wild-type) feeding behavior (Figure 1A; Liu et al., 2013). These data suggest that intestinal INX-16 function contributes to the animal's feeding behaviors.
Having shown that inx-16's actions in the intestine can rescue the social feeding phenotype, we queried whether aggregated feeding was a common feature of mutants with impaired digestive waste release (Thomas, 1990). Defecation mutants induce constipation. One constipated mutant with defects in the expulsion step, exp-1, disrupts an excitatory GABA-ergic receptor in enteric muscles and has been shown to mildly increase social feeding behavior (Beg and Jorgensen, 2003; Bendesky et al., 2012). We assayed this mutant, and others, selecting a set of mutants that affect each defecation motor step and all tissue types involved in the motor program (Branicky and Hekimi, 2006; Thomas, 1990). Two genes required for the first motor step (the posterior body contraction), the intestinal sodium proton exchanger, pbo-4/nhx-7, and the downstream proton-gated ion channel, pbo-5, localized to body wall muscles, were tested (Beg et al., 2008; Pfeiffer et al., 2008). Neither of these Pbo mutants showed increases in social feeding in comparison to N2 (Figure 1B). The later steps of the program, anterior body contraction and enteric muscle contraction, require NLP-40 release from the intestine to stimulate GABA release from enteric neurons, AVL and DVB (Wang et al., 2013). NLP-40 binding activates AEX-2, a g-protein coupled receptor, on these neurons (Allman et al., 2016; Mahoney et al., 2008). Like the Pbo mutants, aex-2(sa3) showed no significant increase over wild-type in our aggregation assay (Figure 1B). In our assays, exp-1(sa6) did not increase aggregation, which differs from prior findings that reported a small but significant increase (Bendesky et al., 2012). Though our defecation mutant panel is not exhaustive, these data suggest that the social feeding phenotype of inx-16 is not broadly shared with mutants suffering from constipation; disrupting defecation per se does not alter social feeding behavior in C. elegans.
Two key gene pathways control the choice between solitary and social feeding behavior: npr-1 and daf-7. Therefore, we used genetic assays to elucidate potential connections between inx-16 and these known pathways (de Bono and Bargmann, 1998; de Bono et al., 2002). Double mutants of inx-16 with daf-7(e1372) and npr-1(ad609) were constructed and tested alongside single mutants to determine whether the double mutant lines had enhanced aggregation. The combination of inx-16 and daf-7(e1372) enhanced aggregation substantially, with the inx-16;daf-7 double mutant exhibiting increased aggregation compared to the daf-7 mutant alone, approximately 23% for the double compared to ~13% for either single mutants (Figure 1C). This additive effect is consistent with inx-16 functioning at least partly independently of daf-7. By contrast, the combination of inx-16 and the strong loss of function npr-1 allele, ad609, did not augment the social feeding level relative to that of npr-1 alone. The inx-16;npr-1 double mutants exhibit no increase in aggregation compared to the npr-1 mutant alone (60% for the double compared to 58% for the single mutant, Figure 1C). The results of a two-way ANOVA analysis support a highly significant genetic interaction between inx-16 and npr-1 (P < 0.003; F = (1, 20)=19). These findings are consistent with inx-16 functioning via the npr-1 pathway to mediate social feeding behavior.
Since our findings placed inx-16 within the npr-1 pathway rather than the daf-7 pathway, we searched for NPR-1 ligands with evidence of intestinal expression. Of the two identified NPR-1 ligands, FLP-18 and FLP-21, flp-21 showed intestinal expression in addition to a subset of neurons (Kubiak et al., 2003; Rogers et al., 2003). The flp-21 mutation causes a significantly increased propensity for animals to aggregate, enhancing aggregation to ~15% (Figure 1D). Interestingly, flp-21's magnitude of impact on feeding behavior is similar to that of inx-16 mutation, ~15% for flp-21 vs. ~14% for inx-16 (Figure 1D; Rogers et al., 2003). Thus, we hypothesized that FLP-21 could be released from the intestine following calcium waves in a manner analogous to NLP-40 release. To begin to investigate this model, we explored epistasis between inx-16 and flp-21. The double mutant line exhibited an aggregation profile very similar to that of either individual mutant strain (inx-16;flp-21 = ~ 13%; Figure 1D). Statistical analysis for gene interaction upholds this epistatic relationship (P < 0.0001; F = (1, 28) = 89). Our results support a model placing FLP-21 release downstream of INX-16 function, and by inference the rhythmic intestinal calcium wave that directs the digestive motor program (Dal Santo et al., 1999; Espelt et al., 2005; Teramoto and Iwasaki, 2006).
Due to flp-21's reported intestinal localization, we suggest that inx-16's social feeding phenotype may be due to reduced FLP-21 release from the intestine. Since NPR-1 signaling dampens social feeding, loss of FLP-21 would increase aggregation, as seen in both inx-16 and flp-21 mutants. Our model is supported by the previously reported epistasis analysis of flp-21(pk1601) and npr-1(ad609); this flp-21 allele did not enhance npr-1(ad609)'s social feeding level, matching our results (Figure 1C; Rogers et al., 2003). Given that flp-18, the other known NPR-1 ligand, is not expressed in the intestine, flp-18 presumably remains active in the inx-16 mutant, allowing ample residual suppression of social feeding to result in the modest aggregation seen in both the inx-16 and flp-21 mutants (Figure 1D; Kapahi et al., 2010).
In summary, our findings demonstrate a behavioral feeding phenotype associated with disrupted intestinal calcium wave dynamics. We propose that this phenotype is due to disruption of FLP-21 mediated activation of NPR-1. Moreover, the social feeding phenotype of inx-16 suggests that the periodic intestinal calcium waves serve to regulate peptide release broadly, influencing a host of behaviors beyond defecation and adding to the growing body of evidence illustrating the importance of gut-to-brain signaling for animal behavior.
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","pubmedId":"","doi":"10.1016/j.cub.2013.03.049"}],"title":"Gap junctional communication in the intestine affects social feeding behavior via FLP-21 and the NPR-1 mediated signaling pathway
","reviews":[],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":"1780344608021"}]},{"id":"306eeab8-bfb3-42d3-9f2f-d96bcd9c0d4c","decision":"edit","abstract":"Communication between the gut and the nervous system coordinates aspects of behavior and responses to stressors. When actively feeding, C. elegans executes a digestive motor program triggered by a calcium wave that requires gap junctional connections along the intestinal length. Loss of function of the innexin-16 (inx-16) gap junctional subunit results in altered group feeding behavior, leading to social feeding characterized by increased body contact, or clumping, between animals. Using genetic analysis, our data shows that inx-16 genetically interacts with npr-1 and flp-21, but not daf-7. Thus, gut-to-brain signaling influences social versus solitary feeding behavioral choice.
","acknowledgements":"We thank the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), for several strains, and the National BioResource Project (NBRP) which is funded by the Japanese government for the inx-16 (tm1589) mutant, and the Zhao-Wen Wang lab for sharing a strain. Taylor Allen's generous sharing of equipment and Amy Vashlishan Murray's helpful insights and fruitful discussions are also appreciated.
","authors":[{"affiliations":["IQ Solutions, Inc, Rockville, MD, USA "],"departments":["Science Communications"],"credit":["formalAnalysis","investigation","writing_reviewEditing","methodology","conceptualization"],"email":"learmanlisa@gmail.com","firstName":"Lisa ","lastName":"Calabro","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Oberlin College"],"departments":["Biology"],"credit":["conceptualization","formalAnalysis","project","supervision","methodology","visualization","writing_originalDraft"],"email":"mpeters@oberlin.edu","firstName":"Maureen","lastName":"Peters","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0001-0337-0790"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by the Oberlin College Biology Department.
","image":{"url":"https://portal.micropublication.org/uploads/05794fc712c685f84ac659d5bd0dca96.jpeg"},"imageCaption":"Figure 1: inx-16 loss of function increases aggregation, and genetic interaction assays show an interaction between flp-21 and npr-1 but not daf-7. (A) Aggregation fractions for N2; npr-1(ad609); inx-16(tm1589) mutant with inx-16 (+) expressed in the muscle (mus.) and inx-16 mutant with inx-16 (+) expressed in the adult intestinal (int.), both labeled as + “rescue”, n=9 for all strains. (B) Aggregation fractions for defecation mutants compared to N2, mutants used: aex-2(sa3); exp-1(sa6); pbo-4(ok583); pbo-5(ox4); n=6 for all strains. (C) Aggregation fractions for single and double mutants of the following genes: inx-16(tm1589); npr-1(ad609); daf-7(e1372); n=6 for all strains. (D) Aggregation fractions for single and double mutants of the following genes: flp-21(ok889); inx-16(tm1589) alongside N2; n=8 for all strains. Statistical significance: *p<0.05; **= p<0.01; ***<0.001; ****<0.0001; ns is not significant. Brackets used for comparisons between two mutants; line used across three strains for genetic interactions.
","imageTitle":"inx-16 mutation results in enhanced aggregation and genetically interacts with both nlp-1 and flp-21
","methods":"Strain maintenance and construction
C. elegans strains were cultured on standard NGM plates seeded with OP50 bacteria using standard protocols. Double daf-2 and npr-1 mutant strains were generated using standard genetic crossing, selection based on dauer or aggregation phenotype, followed by sequencing to confirm genotypes. For flp-21, the large deletion allowed the use of PCR tracking prior to sequencing.
Social feeding assay
Young adult animals were assayed blindly using a protocol adapted from De Bono and Bargmann (1998). Strains containing the daf-2(e1372) mutation were reared at 15o C to prevent dauer formation. NGM plates were seeded with 200 mL of OP50 in a circular lawn about 25 mm in diameter. Approximately 150 well-fed day 1 adult worms were added and left at 20-22o C for 2 hours. Then, the fraction of animals in contact with two or more other animals along at least 50% of their body length was tallied by visual inspection. For each panel's experiments, the full set of strains was tested each assay day and the full set of assays were completed in a short time period.
Statistical analysis and graphing
For statistical analysis, Graphpad/Prism (Version 11 for macOS) was used. Each panel's data set was tested for Gaussian distribution. Only one panel's data set (A) showed some non-Gaussian distribution, so Kruskal-Wallis program was applied. Otherwise, ANOVA analysis followed by multiple comparisons using Tukey's test (one-way ANOVA for panels A and B; two-way ANOVA for panels C and D) was used. Statistical deviation was considered significant if p<0.05. Bar graphs show all data points, mean and standard error of the mean. p value scores are listed for informative comparisons relevant to our study. Differences between npr-1 and N2, or other mutants that were previously published and/or strikingly obvious were not specifically noted. For example, statistical tests between N2 and npr-1; inx-16 and npr-1; npr-1 and daf-7 were not highlighted to simplify the figures.
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inx-16(tm1589) I; zwEx701[pmyo-3::inx-16(+)::GFP + pmyo-3::GFP] | Zhao-Wen Wang Lab |
Organisms adapt to their dynamic internal and external environments by leveraging systemic physiological responses using signaling pathways involving multiple tissues and extensive cross-talk. Recently, the gut has emerged as an important signaling center keenly attuned to an animal's feeding state, food quality, and pathogenic exposure. Within C. elegans, gut-derived secreted peptide signals influence feeding, stress response, waste release and avoidance behavior by acting on neurons of the enteric and central nervous systems (Artan et al., 2016; Jia et al., 2024; Sural et al., 2025). For example, INS-7 and INS-35 regulate the NSM neuron to adjust pharyngeal pumping rates (Sural et al., 2025). Additional insulin family members such as INS-3 and INS-11 function in gut-to-brain signaling pathways (Lee and Mylonakis, 2017; Veuthey et al., 2025). Several non-insulin peptides are also involved. FLP-2 (FMRFamide-like peptide) acts via the interneuron AIY to induce responses to oxidative stress within the animal (Jia et al., 2024). NLP-40 (Neuropeptide-like protein) is released to instruct the posterior enteric neurons, AVL and DVB, to elicit steps of the digestive/defecation motor program; its release is tied to a periodic calcium wave that occurs in well-fed animals with a regular frequency of 45-55 seconds (Choi et al., 2023; Mahoney et al., 2008; Wang et al., 2013).
This regular calcium wave may trigger release of additional peptides that influence nervous system behavior more broadly. Several mutants with poor digestive waste release originally identified due to their constipated phenotypes turn out to encode integral components of peptide processing and release such as egl-3, egl-21, aex-1, aex-4, and aex-5 (Husson et al., 2007; Khawand et al., 2026; Mahoney et al., 2008). At least one peptide, NLP-40, is released in time with the digestive motor program, via activation of synaptotagmin-2 (snt-2), a calcium-sensing protein that triggers DCV fusion (Wang et al., 2013). The tyramine response pathway offers additional evidence of calcium-triggered peptide release (Veuthey et al., 2025). This enticing evidence suggests that the rhythmic calcium flux that triggers NLP-40 release, and other steps of the digestive/defecation motor program, may be used more broadly to influence behavioral choices where feeding and nutritional status are relevant. With this notion in mind, we explored an observation of social feeding behavior in a mutant that alters intestinal calcium wave dynamics.
The innexin-16 (inx-16) gap junction subunit is critical for periodic intestinal calcium waves associated with the digestive motor program; loss of function inx-16 mutants disrupt the intestinal calcium wave and greatly reduce the digestive motor program steps that require NLP-40 release (Peters et al., 2007). During routine maintenance of inx-16(tm1589), we observed increased aggregation of well-fed worms and became intrigued. To explore this further, we assayed for social feeding behavior quantitatively. For comparison, we included N2, a solitary strain, and a social feeding strain, npr-1(ad609). The gene, npr-1, encodes a C. elegans receptor related to neuropeptide Y and is neuronally expressed. When active, NPR-1 inhibits the tendency of animals to aggregate in groups, suppresses oxygen level preferences, and affects bordering (de Bono and Bargmann, 1998; Gray et al., 2004). The npr-1 allele we used, ad609, has been characterized to be a strong loss of function allele, equivalent to a genetic null (de Bono and Bargmann, 1998). Our results show that inx-16 mutant animals maintain contact with other animals more frequently than wild-type (N2) solitary worms; young adult inx-16(tm1589) animals demonstrated a significant difference in the number of inx-16 animals found in direct contact with their peers compared to N2 (~0% vs. ~15%; Figure 1A). Yet, the inx-16 phenotype is much weaker than the npr-1(ad609) allele (~80% vs. ~15%; Figure 1A).
To verify that INX-16 functions within the intestine to cause the observed tendency to aggregate, we employed tissue specific rescue of inx-16 mutants. Our and others' inx-16 expression analyses indicated that inx-16 was only expressed in the intestine, yet two studies have provided phenotypic evidence suggesting additional areas of function (Bhattacharya et al., 2019; Liu et al., 2013; Sojka et al., 2025). Intestine-specific rescue was achieved using a construct with a short, well-defined fragment, regulatory sequence of the egg yolk protein, vitellogenin-2, (vit-2) gene, placed upstream of an INX-16 translational fusion (MacMorris et al., 1992). Prior work using this construct led to full rescue of the defecation defects in adults (Peters et al., 2007). Likewise, solitary feeding behavior was restored in the inx-16+pvit-2 intestinal rescue line (Figure 1A). By contrast, driving expression of inx-16 in body wall muscles, using the myo-3 promoter, was unable to restore solitary (wild-type) feeding behavior (Figure 1A; Liu et al., 2013). These data suggest that intestinal INX-16 function contributes to the animal's feeding behaviors.
Having shown that inx-16's actions in the intestine can rescue the social feeding phenotype, we queried whether aggregated feeding was a common feature of mutants with impaired digestive waste release (Thomas, 1990). Defecation mutants induce constipation. One constipated mutant with defects in the expulsion step, exp-1, disrupts an excitatory GABA-ergic receptor in enteric muscles and has been shown to mildly increase social feeding behavior (Beg and Jorgensen, 2003; Bendesky et al., 2012). We assayed this mutant, and others, selecting a set of mutants that affect each defecation motor step and all tissue types involved in the motor program (Branicky and Hekimi, 2006; Thomas, 1990). Two genes required for the first motor step (the posterior body contraction), the intestinal sodium proton exchanger, pbo-4/nhx-7, and the downstream proton-gated ion channel, pbo-5, localized to body wall muscles, were tested (Beg et al., 2008; Pfeiffer et al., 2008). Neither of these Pbo mutants showed increases in social feeding in comparison to N2 (Figure 1B). The later steps of the program, anterior body contraction and enteric muscle contraction, require NLP-40 release from the intestine to stimulate GABA release from enteric neurons, AVL and DVB (Wang et al., 2013). NLP-40 binding activates AEX-2, a g-protein coupled receptor, on these neurons (Allman et al., 2016; Mahoney et al., 2008). Like the Pbo mutants, aex-2(sa3) showed no significant increase over wild-type in our aggregation assay (Figure 1B). In our assays, exp-1(sa6) did not increase aggregation, which differs from prior findings that reported a small but significant increase (Bendesky et al., 2012). Though our defecation mutant panel is not exhaustive, these data suggest that the social feeding phenotype of inx-16 is not broadly shared with mutants suffering from constipation; disrupting defecation per se does not alter social feeding behavior in C. elegans.
Two key gene pathways control the choice between solitary and social feeding behavior: npr-1 and daf-7. Therefore, we used genetic assays to elucidate potential connections between inx-16 and these known pathways (de Bono and Bargmann, 1998; de Bono et al., 2002). Double mutants of inx-16 with daf-7(e1372) and npr-1(ad609) were constructed and tested alongside single mutants to determine whether the double mutant lines had enhanced aggregation. The combination of inx-16 and daf-7(e1372) enhanced aggregation substantially, with the inx-16;daf-7 double mutant exhibiting increased aggregation compared to the daf-7 mutant alone, approximately 23% for the double compared to ~13% for either single mutants (Figure 1C). This additive effect is consistent with inx-16 functioning at least partly independently of daf-7. By contrast, the combination of inx-16 and the strong loss of function npr-1 allele, ad609, did not augment the social feeding level relative to that of npr-1 alone. The inx-16;npr-1 double mutants exhibit no increase in aggregation compared to the npr-1 mutant alone (60% for the double compared to 58% for the single mutant, Figure 1C). The results of a two-way ANOVA analysis support a highly significant genetic interaction between inx-16 and npr-1 (P < 0.003; F = (1, 20)=19). These findings are consistent with inx-16 functioning via the npr-1 pathway to mediate social feeding behavior.
Since our findings placed inx-16 within the npr-1 pathway rather than the daf-7 pathway, we searched for NPR-1 ligands with evidence of intestinal expression. Of the two identified NPR-1 ligands, FLP-18 and FLP-21, flp-21 showed intestinal expression in addition to a subset of neurons (Kubiak et al., 2003; Rogers et al., 2003). The flp-21 mutation causes a significantly increased propensity for animals to aggregate, enhancing aggregation to ~15% (Figure 1D). Interestingly, flp-21's magnitude of impact on feeding behavior is similar to that of inx-16 mutation, ~15% for flp-21 vs. ~14% for inx-16 (Figure 1D; Rogers et al., 2003). Thus, we hypothesized that FLP-21 could be released from the intestine following calcium waves in a manner analogous to NLP-40 release. To begin to investigate this model, we explored epistasis between inx-16 and flp-21. The double mutant line exhibited an aggregation profile very similar to that of either individual mutant strain (inx-16;flp-21 = ~ 13%; Figure 1D). Statistical analysis for gene interaction upholds this epistatic relationship (P < 0.0001; F = (1, 28) = 89). Our results support a model placing FLP-21 release downstream of INX-16 function, and by inference the rhythmic intestinal calcium wave that directs the digestive motor program (Dal Santo et al., 1999; Espelt et al., 2005; Teramoto and Iwasaki, 2006).
Due to flp-21's reported intestinal localization, we suggest that inx-16's social feeding phenotype may be due to reduced FLP-21 release from the intestine. Since NPR-1 signaling dampens social feeding, loss of FLP-21 would increase aggregation, as seen in both inx-16 and flp-21 mutants. Our model is supported by the previously reported epistasis analysis of flp-21(pk1601) and npr-1(ad609); this flp-21 allele did not enhance npr-1(ad609)'s social feeding level, matching our results (Figure 1C; Rogers et al., 2003). Given that flp-18, the other known NPR-1 ligand, is not expressed in the intestine, flp-18 presumably remains active in the inx-16 mutant, allowing ample residual suppression of social feeding to result in the modest aggregation seen in both the inx-16 and flp-21 mutants (Figure 1D; Kapahi et al., 2010).
In summary, our findings demonstrate a behavioral feeding phenotype associated with disrupted intestinal calcium wave dynamics. We propose that this phenotype is due to disruption of FLP-21 mediated activation of NPR-1. Moreover, the social feeding phenotype of inx-16 suggests that the periodic intestinal calcium waves serve to regulate peptide release broadly, influencing a host of behaviors beyond defecation and adding to the growing body of evidence illustrating the importance of gut-to-brain signaling for animal behavior.
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","pubmedId":"","doi":"10.1016/j.cub.2013.03.049"}],"title":"Gap junctional communication in the intestine affects social feeding behavior via FLP-21 and the NPR-1 mediated signaling pathway
","reviews":[],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":null}]},{"id":"9c3850ba-08db-4e43-a66e-8fa2da473802","decision":"edit","abstract":"Communication between the gut and the nervous system coordinates aspects of behavior and responses to stressors. When actively feeding, C. elegans executes a digestive motor program triggered by a calcium wave that requires gap junctional connections along the intestinal length. Loss of function of the innexin-16 (inx-16) gap junctional subunit results in altered group feeding behavior, leading to social feeding characterized by increased body contact, or clumping, between animals. Using genetic analysis, our data shows that inx-16 genetically interacts with npr-1 and flp-21, but not daf-7. Thus, gut-to-brain signaling influences social versus solitary feeding behavioral choice.
","acknowledgements":"We thank the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), for several strains, and the National BioResource Project (NBRP) which is funded by the Japanese government for the inx-16 (tm1589) mutant, and the Zhao-Wen Wang lab for sharing a strain. Taylor Allen's generous sharing of equipment and Amy Vashlishan Murray's helpful insights and fruitful discussions are also appreciated.
","authors":[{"affiliations":["IQ Solutions, Inc, Rockville, MD, USA "],"departments":["Science Communications"],"credit":["formalAnalysis","investigation","writing_reviewEditing","methodology","conceptualization"],"email":"learmanlisa@gmail.com","firstName":"Lisa ","lastName":"Calabro","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Oberlin College"],"departments":["Biology"],"credit":["conceptualization","formalAnalysis","project","supervision","methodology","visualization","writing_originalDraft"],"email":"mpeters@oberlin.edu","firstName":"Maureen","lastName":"Peters","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0001-0337-0790"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by the Oberlin College Biology Department.
","image":{"url":"https://portal.micropublication.org/uploads/05794fc712c685f84ac659d5bd0dca96.jpeg"},"imageCaption":"Figure 1: inx-16 loss of function increases aggregation, and genetic interaction assays show an interaction between flp-21 and npr-1 but not daf-7. (A) Aggregation fractions for N2; npr-1(ad609); inx-16(tm1589) mutant with inx-16 (+) expressed in the muscle (mus.) and inx-16 mutant with inx-16 (+) expressed in the adult intestinal (int.), both labeled as + “rescue”, n=9 for all strains. (B) Aggregation fractions for defecation mutants compared to N2, mutants used: aex-2(sa3); exp-1(sa6); pbo-4(ok583); pbo-5(ox4); n=6 for all strains. (C) Aggregation fractions for single and double mutants of the following genes: inx-16(tm1589); npr-1(ad609); daf-7(e1372); n=6 for all strains. (D) Aggregation fractions for single and double mutants of the following genes: flp-21(ok889); inx-16(tm1589) alongside N2; n=8 for all strains. Statistical significance: *p<0.05; **= p<0.01; ***<0.001; ****<0.0001; ns is not significant. Brackets used for comparisons between two mutants; line used across three strains for genetic interactions.
","imageTitle":"inx-16 mutation results in enhanced aggregation and genetically interacts with both nlp-1 and flp-21
","methods":"Strain maintenance and construction
C. elegans strains were cultured on standard NGM plates seeded with OP50 bacteria using standard protocols. Double daf-2 and npr-1 mutant strains were generated using standard genetic crossing, selection based on dauer or aggregation phenotype, followed by sequencing to confirm genotypes. For flp-21, the large deletion allowed the use of PCR tracking prior to sequencing.
Social feeding assay
Young adult animals were assayed blindly using a protocol adapted from De Bono and Bargmann (1998). Strains containing the daf-2(e1372) mutation were reared at 15o C to prevent dauer formation. NGM plates were seeded with 200 mL of OP50 in a circular lawn about 25 mm in diameter. Approximately 150 well-fed day 1 adult worms were added and left at 20-22o C for 2 hours. Then, the fraction of animals in contact with two or more other animals along at least 50% of their body length was tallied by visual inspection. For each panel's experiments, the full set of strains was tested each assay day and the full set of assays were completed in a short time period.
Statistical analysis and graphing
For statistical analysis, Graphpad/Prism (Version 11 for macOS) was used. Each panel's data set was tested for Gaussian distribution. Only one panel's data set (A) showed some non-Gaussian distribution, so Kruskal-Wallis program was applied. Otherwise, ANOVA analysis followed by multiple comparisons using Tukey's test (one-way ANOVA for panels A and B; two-way ANOVA for panels C and D) was used. Statistical deviation was considered significant if p<0.05. Bar graphs show all data points, mean and standard error of the mean. p value scores are listed for informative comparisons relevant to our study. Differences between npr-1 and N2, or other mutants that were previously published and/or strikingly obvious were not specifically noted. For example, statistical tests between N2 and npr-1; inx-16 and npr-1; npr-1 and daf-7 were not highlighted to simplify the figures.
","reagents":"Strain | Genotype | Available from |
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inx-16(tm1589) I; zwEx701[pmyo-3::inx-16(+)::GFP + pmyo-3::GFP] | Zhao-Wen Wang Lab |
Organisms adapt to their dynamic internal and external environments by leveraging systemic physiological responses using signaling pathways involving multiple tissues and extensive cross-talk. Recently, the gut has emerged as an important signaling center keenly attuned to an animal's feeding state, food quality, and pathogenic exposure. Within C. elegans, gut-derived secreted peptide signals influence feeding, stress response, waste release and avoidance behavior by acting on neurons of the enteric and central nervous systems (Artan et al., 2016; Jia et al., 2024; Sural et al., 2025). For example, INS-7 and INS-35 regulate the NSM neuron to adjust pharyngeal pumping rates (Sural et al., 2025). Additional insulin family members such as INS-3 and INS-11 function in gut-to-brain signaling pathways (Lee and Mylonakis, 2017; Veuthey et al., 2025). Several non-insulin peptides are also involved. FLP-2 (FMRFamide-like peptide) acts via the interneuron AIY to induce responses to oxidative stress within the animal (Jia et al., 2024). NLP-40 (Neuropeptide-like protein) is released to instruct the posterior enteric neurons, AVL and DVB, to elicit steps of the digestive/defecation motor program; its release is tied to a periodic calcium wave that occurs in well-fed animals with a regular frequency of 45-55 seconds (Choi et al., 2023; Mahoney et al., 2008; Wang et al., 2013).
This regular calcium wave may trigger release of additional peptides that influence nervous system behavior more broadly. Several mutants with poor digestive waste release originally identified due to their constipated phenotypes turn out to encode integral components of peptide processing and release such as egl-3, egl-21, aex-1, aex-4, and aex-5 (Husson et al., 2007; Khawand et al., 2026; Mahoney et al., 2008). At least one peptide, NLP-40, is released in time with the digestive motor program, via activation of synaptotagmin-2 (snt-2), a calcium-sensing protein that triggers DCV fusion (Wang et al., 2013). The tyramine response pathway offers additional evidence of calcium-triggered peptide release (Veuthey et al., 2025). This enticing evidence suggests that the rhythmic calcium flux that triggers NLP-40 release, and other steps of the digestive/defecation motor program, may be used more broadly to influence behavioral choices where feeding and nutritional status are relevant. With this notion in mind, we explored an observation of social feeding behavior in a mutant that alters intestinal calcium wave dynamics.
The innexin-16 (inx-16) gap junction subunit is critical for periodic intestinal calcium waves associated with the digestive motor program; loss of function inx-16 mutants disrupt the intestinal calcium wave and greatly reduce the digestive motor program steps that require NLP-40 release (Peters et al., 2007). During routine maintenance of inx-16(tm1589), we observed increased aggregation of well-fed worms and became intrigued. To explore this further, we assayed for social feeding behavior quantitatively. For comparison, we included N2, a solitary strain, and a social feeding strain, npr-1(ad609). The gene, npr-1, encodes a C. elegans receptor related to neuropeptide Y and is neuronally expressed. When active, NPR-1 inhibits the tendency of animals to aggregate in groups, suppresses oxygen level preferences, and affects bordering (de Bono and Bargmann, 1998; Gray et al., 2004). The npr-1 allele we used, ad609, has been characterized to be a strong loss of function allele, equivalent to a genetic null (de Bono and Bargmann, 1998). Our results show that inx-16 mutant animals maintain contact with other animals more frequently than wild-type (N2) solitary worms; young adult inx-16(tm1589) animals demonstrated a significant difference in the number of inx-16 animals found in direct contact with their peers compared to N2 (~0% vs. ~15%; Figure 1A). Yet, the inx-16 phenotype is much weaker than the npr-1(ad609) allele (~80% vs. ~15%; Figure 1A).
To verify that INX-16 functions within the intestine to cause the observed tendency to aggregate, we employed tissue specific rescue of inx-16 mutants. Our and others' inx-16 expression analyses indicated that inx-16 was only expressed in the intestine, yet two studies have provided phenotypic evidence suggesting additional areas of function (Bhattacharya et al., 2019; Liu et al., 2013; Sojka et al., 2025). Intestine-specific rescue was achieved using a construct with a short, well-defined fragment, regulatory sequence of the egg yolk protein, vitellogenin-2, (vit-2) gene, placed upstream of an INX-16 translational fusion (MacMorris et al., 1992). Prior work using this construct led to full rescue of the defecation defects in adults (Peters et al., 2007). Likewise, solitary feeding behavior was restored in the inx-16+pvit-2 intestinal rescue line (Figure 1A). By contrast, driving expression of inx-16 in body wall muscles, using the myo-3 promoter, was unable to restore solitary (wild-type) feeding behavior (Figure 1A; Liu et al., 2013). These data suggest that intestinal INX-16 function contributes to the animal's feeding behaviors.
Having shown that inx-16's actions in the intestine can rescue the social feeding phenotype, we queried whether aggregated feeding was a common feature of mutants with impaired digestive waste release (Thomas, 1990). Defecation mutants induce constipation. One constipated mutant with defects in the expulsion step, exp-1, disrupts an excitatory GABA-ergic receptor in enteric muscles and has been shown to mildly increase social feeding behavior (Beg and Jorgensen, 2003; Bendesky et al., 2012). We assayed this mutant, and others, selecting a set of mutants that affect each defecation motor step and all tissue types involved in the motor program (Branicky and Hekimi, 2006; Thomas, 1990). Two genes required for the first motor step (the posterior body contraction), the intestinal sodium proton exchanger, pbo-4/nhx-7, and the downstream proton-gated ion channel, pbo-5, localized to body wall muscles, were tested (Beg et al., 2008; Pfeiffer et al., 2008). Neither of these Pbo mutants showed increases in social feeding in comparison to N2 (Figure 1B). The later steps of the program, anterior body contraction and enteric muscle contraction, require NLP-40 release from the intestine to stimulate GABA release from enteric neurons, AVL and DVB (Wang et al., 2013). NLP-40 binding activates AEX-2, a g-protein coupled receptor, on these neurons (Allman et al., 2016; Mahoney et al., 2008). Like the Pbo mutants, aex-2(sa3) showed no significant increase over wild-type in our aggregation assay (Figure 1B). In our assays, exp-1(sa6) did not increase aggregation, which differs from prior findings that reported a small but significant increase (Bendesky et al., 2012). Though our defecation mutant panel is not exhaustive, these data suggest that the social feeding phenotype of inx-16 is not broadly shared with mutants suffering from constipation; disrupting defecation per se does not alter social feeding behavior in C. elegans.
Two key gene pathways control the choice between solitary and social feeding behavior: npr-1 and daf-7. Therefore, we used genetic assays to elucidate potential connections between inx-16 and these known pathways (de Bono and Bargmann, 1998; de Bono et al., 2002). Double mutants of inx-16 with daf-7(e1372) and npr-1(ad609) were constructed and tested alongside single mutants to determine whether the double mutant lines had enhanced aggregation. The combination of inx-16 and daf-7(e1372) enhanced aggregation substantially, with the inx-16;daf-7 double mutant exhibiting increased aggregation compared to the daf-7 mutant alone, approximately 23% for the double compared to ~13% for either single mutants (Figure 1C). This additive effect is consistent with inx-16 functioning at least partly independently of daf-7. By contrast, the combination of inx-16 and the strong loss of function npr-1 allele, ad609, did not augment the social feeding level relative to that of npr-1 alone. The inx-16;npr-1 double mutants exhibit no increase in aggregation compared to the npr-1 mutant alone (60% for the double compared to 58% for the single mutant, Figure 1C). The results of a two-way ANOVA analysis support a highly significant genetic interaction between inx-16 and npr-1 (P < 0.003; F = (1, 20)=19). These findings are consistent with inx-16 functioning via the npr-1 pathway to mediate social feeding behavior.
Since our findings placed inx-16 within the npr-1 pathway rather than the daf-7 pathway, we searched for NPR-1 ligands with evidence of intestinal expression. Of the two identified NPR-1 ligands, FLP-18 and FLP-21, flp-21 showed intestinal expression in addition to a subset of neurons (Kubiak et al., 2003; Rogers et al., 2003). The flp-21 mutation causes a significantly increased propensity for animals to aggregate, enhancing aggregation to ~15% (Figure 1D). Interestingly, flp-21's magnitude of impact on feeding behavior is similar to that of inx-16 mutation, ~15% for flp-21 vs. ~14% for inx-16 (Figure 1D; Rogers et al., 2003). Thus, we hypothesized that FLP-21 could be released from the intestine following calcium waves in a manner analogous to NLP-40 release. To begin to investigate this model, we explored epistasis between inx-16 and flp-21. The double mutant line exhibited an aggregation profile very similar to that of either individual mutant strain (inx-16;flp-21 = ~ 13%; Figure 1D). Statistical analysis for gene interaction upholds this epistatic relationship (P < 0.0001; F = (1, 28) = 89). Our results support a model placing FLP-21 release downstream of INX-16 function, and by inference the rhythmic intestinal calcium wave that directs the digestive motor program (Dal Santo et al., 1999; Espelt et al., 2005; Teramoto and Iwasaki, 2006).
Due to flp-21's reported intestinal localization, we suggest that inx-16's social feeding phenotype may be due to reduced FLP-21 release from the intestine. Since NPR-1 signaling dampens social feeding, loss of FLP-21 would increase aggregation, as seen in both inx-16 and flp-21 mutants. Our model is supported by the previously reported epistasis analysis of flp-21(pk1601) and npr-1(ad609); this flp-21 allele did not enhance npr-1(ad609)'s social feeding level, matching our results (Figure 1C; Rogers et al., 2003). Given that flp-18, the other known NPR-1 ligand, is not expressed in the intestine, flp-18 presumably remains active in the inx-16 mutant, allowing ample residual suppression of social feeding to result in the modest aggregation seen in both the inx-16 and flp-21 mutants (Figure 1D; Kapahi et al., 2010).
In summary, our findings demonstrate a behavioral feeding phenotype associated with disrupted intestinal calcium wave dynamics. We propose that this phenotype is due to disruption of FLP-21 mediated activation of NPR-1. Moreover, the social feeding phenotype of inx-16 suggests that the periodic intestinal calcium waves serve to regulate peptide release broadly, influencing a host of behaviors beyond defecation and adding to the growing body of evidence illustrating the importance of gut-to-brain signaling for animal behavior.
","references":[{"reference":"Allman E, Wang Q, Walker RL, Austen M, Peters MA, Nehrke K. 2016. Calcineurin homologous proteins regulate the membrane localization and activity of sodium/proton exchangers in C. elegans. American Journal of Physiology-Cell Physiology 310: C233-C242.
","pubmedId":"","doi":"10.1152/ajpcell.00291.2015"},{"reference":"Artan M, Jeong DE, Lee D, Kim YI, Son HG, Husain Z, et al., Lee. 2016. Food-derived sensory cues modulate longevity via distinct neuroendocrine insulin-like peptides. Genes & Development 30: 1047-1057.
","pubmedId":"","doi":"10.1101/gad.279448.116"},{"reference":"Beg AA, Ernstrom GG, Nix P, Davis MW, Jorgensen EM. 2008. Protons Act as a Transmitter for Muscle Contraction in C. elegans. Cell 132: 149-160.
","pubmedId":"","doi":"10.1016/j.cell.2007.10.058"},{"reference":"Beg AA, Jorgensen EM. 2003. EXP-1 is an excitatory GABA-gated cation channel. Nature Neuroscience 6: 1145-1152.
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","pubmedId":"","doi":"10.1371/journal.pgen.1003157"},{"reference":"Bhattacharya A, Aghayeva U, Berghoff EG, Hobert O. 2019. Plasticity of the Electrical Connectome of C. elegans. Cell 176: 1174-1189.e16.
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","pubmedId":"","doi":"10.1016/j.tig.2006.08.006"},{"reference":"Choi U, Hu M, Zhang Q, Sieburth D. 2023. The head mesodermal cell couples FMRFamide neuropeptide signaling with rhythmic muscle contraction in C. elegans. Nature Communications 14, 4218 (2023).
","pubmedId":"","doi":"10.1038/s41467-023-39955-8"},{"reference":"Dal Santo P, Logan MA, Chisholm AD, Jorgensen EM. 1999. The Inositol Trisphosphate Receptor Regulates a 50-Second Behavioral Rhythm in C. elegans. Cell 98: 757-767.
","pubmedId":"","doi":"10.1016/s0092-8674(00)81510-x"},{"reference":"de Bono M, Bargmann CI. 1998. Natural Variation in a Neuropeptide Y Receptor Homolog Modifies Social Behavior and Food Response in C. elegans. Cell 94: 679-689.
","pubmedId":"9741632","doi":"10.1016/s0092-8674(00)81609-8"},{"reference":"de Bono M, Tobin DM, Davis MW, Avery L, Bargmann CI. 2002. Social feeding in Caenorhabditis elegans is induced by neurons that detect aversive stimuli. Nature 419: 899-903.
","pubmedId":"","doi":"10.1038/nature01169"},{"reference":"Espelt MV, Estevez AY, Yin X, Strange K. 2005. Oscillatory Ca2+ Signaling in the IsolatedCaenorhabditis elegansIntestine. The Journal of General Physiology 126: 379-392.
","pubmedId":"","doi":" 10.1085/jgp.200509355"},{"reference":"Gray JM, Karow DS, Lu H, Chang AJ, Chang JS, Ellis RE, Marletta MA, Bargmann CI. 2004. Oxygen sensation and social feeding mediated by a C. elegans guanylate cyclase homologue. Nature 430: 317-322.
","pubmedId":"","doi":"10.1038/nature02714"},{"reference":"Husson SJ, Janssen T, Baggerman G, Bogert B, Kahn‐Kirby AH, Ashrafi K, Schoofs L. 2007. Impaired processing of FLP and NLP peptides in carboxypeptidase E (EGL‐21)‐deficient Caenorhabditis elegans as analyzed by mass spectrometry. Journal of Neurochemistry 102: 246-260.
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","pubmedId":"","doi":"10.1093/genetics/124.4.855"},{"reference":"Veuthey T, Florman JT, Giunti Sn, Romussi S, De Rosa MaJ, Alkema MJ, Rayes D. 2025. The neurohormone tyramine stimulates the secretion of an insulin-like peptide from the Caenorhabditis elegans intestine to modulate the systemic stress response. PLOS Biology 23: e3002997.
","pubmedId":"","doi":"10.1371/journal.pbio.3002997"},{"reference":"Wang H, Girskis K, Janssen T, Chan JP, Dasgupta K, Knowles JA, Schoofs L, Sieburth D. 2013. Neuropeptide Secreted from a Pacemaker Activates Neurons to Control a Rhythmic Behavior. Current Biology 23: 746-754.
","pubmedId":"","doi":"10.1016/j.cub.2013.03.049"}],"title":"Gap junctional communication in the intestine affects social feeding behavior via FLP-21 and the NPR-1 mediated signaling pathway
","reviews":[],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":null}]},{"id":"12187cd1-3ddb-4591-84e3-c56f71df2a7b","decision":"publish","abstract":"Communication between the gut and the nervous system coordinates aspects of behavior and responses to stressors. When actively feeding, C. elegans executes a digestive motor program triggered by a calcium wave that requires gap junctional connections along the intestinal length. Loss of function of the innexin-16 (inx-16) gap junctional subunit results in altered group feeding behavior, leading to social feeding characterized by increased body contact, or clumping, between animals. Using genetic analysis, our data shows that inx-16 genetically interacts with npr-1 and flp-21, but not daf-7. Thus, gut-to-brain signaling influences social versus solitary feeding behavioral choice.
","acknowledgements":"We thank the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), for several strains, and the National BioResource Project (NBRP) which is funded by the Japanese government for the inx-16 (tm1589) mutant, and the Zhao-Wen Wang lab for sharing a strain. Taylor Allen's generous sharing of equipment and Amy Vashlishan Murray's helpful insights and fruitful discussions are also appreciated.
","authors":[{"affiliations":["IQ Solutions, Inc, Rockville, MD, USA "],"departments":["Science Communications"],"credit":["formalAnalysis","investigation","writing_reviewEditing","methodology","conceptualization"],"email":"learmanlisa@gmail.com","firstName":"Lisa ","lastName":"Calabro","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Oberlin College"],"departments":["Biology"],"credit":["conceptualization","formalAnalysis","project","supervision","methodology","visualization","writing_originalDraft"],"email":"mpeters@oberlin.edu","firstName":"Maureen","lastName":"Peters","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0001-0337-0790"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by the Oberlin College Biology Department.
","image":{"url":"https://portal.micropublication.org/uploads/05794fc712c685f84ac659d5bd0dca96.jpeg"},"imageCaption":"Figure 1: inx-16 loss of function increases aggregation, and genetic interaction assays show an interaction between flp-21 and npr-1 but not daf-7. (A) Aggregation fractions for N2; npr-1(ad609); inx-16(tm1589) mutant with inx-16 (+) expressed in the muscle (mus.) and inx-16 mutant with inx-16 (+) expressed in the adult intestinal (int.), both labeled as + “rescue”, n=9 for all strains. (B) Aggregation fractions for defecation mutants compared to N2, mutants used: aex-2(sa3); exp-1(sa6); pbo-4(ok583); pbo-5(ox4); n=6 for all strains. (C) Aggregation fractions for single and double mutants of the following genes: inx-16(tm1589); npr-1(ad609); daf-7(e1372); n=6 for all strains. (D) Aggregation fractions for single and double mutants of the following genes: flp-21(ok889); inx-16(tm1589) alongside N2; n=8 for all strains. Statistical significance: *p<0.05; **= p<0.01; ***<0.001; ****<0.0001; ns is not significant. Brackets used for comparisons between two mutants; line used across three strains for genetic interactions.
","imageTitle":"inx-16 mutation results in enhanced aggregation and genetically interacts with both nlp-1 and flp-21
","methods":"Strain maintenance and construction
C. elegans strains were cultured on standard NGM plates seeded with OP50 bacteria using standard protocols. Double daf-2 and npr-1 mutant strains were generated using standard genetic crossing, selection based on dauer or aggregation phenotype, followed by sequencing to confirm genotypes. For flp-21, the large deletion allowed the use of PCR tracking prior to sequencing.
Social feeding assay
Young adult animals were assayed blindly using a protocol adapted from De Bono and Bargmann (1998). Strains containing the daf-2(e1372) mutation were reared at 15o C to prevent dauer formation. NGM plates were seeded with 200 mL of OP50 in a circular lawn about 25 mm in diameter. Approximately 150 well-fed day 1 adult worms were added and left at 20-22o C for 2 hours. Then, the fraction of animals in contact with two or more other animals along at least 50% of their body length was tallied by visual inspection. For each panel's experiments, the full set of strains was tested each assay day and the full set of assays were completed in a short time period.
Statistical analysis and graphing
For statistical analysis, Graphpad/Prism (Version 11 for macOS) was used. Each panel's data set was tested for Gaussian distribution. Only one panel's data set (A) showed some non-Gaussian distribution, so Kruskal-Wallis program was applied. Otherwise, ANOVA analysis followed by multiple comparisons using Tukey's test (one-way ANOVA for panels A and B; two-way ANOVA for panels C and D) was used. Statistical deviation was considered significant if p<0.05. Bar graphs show all data points, mean and standard error of the mean. p value scores are listed for informative comparisons relevant to our study. Differences between npr-1 and N2, or other mutants that were previously published and/or strikingly obvious were not specifically noted. For example, statistical tests between N2 and npr-1; inx-16 and npr-1; npr-1 and daf-7 were not highlighted to simplify the figures.
","reagents":"Strain | Genotype | Available from |
Bristol isolate | CGC | |
National BioResource Project (NBRP) | ||
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CGC | ||
CGC | ||
CGC | ||
CGC | ||
CGC | ||
CGC | ||
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inx-16(tm1589) I; zwEx701[pmyo-3::inx-16(+)::GFP + pmyo-3::GFP] | Zhao-Wen Wang Lab |
Organisms adapt to their dynamic internal and external environments by leveraging systemic physiological responses using signaling pathways involving multiple tissues and extensive cross-talk. Recently, the gut has emerged as an important signaling center keenly attuned to an animal's feeding state, food quality, and pathogenic exposure. Within C. elegans, gut-derived secreted peptide signals influence feeding, stress response, waste release and avoidance behavior by acting on neurons of the enteric and central nervous systems (Artan et al., 2016; Jia et al., 2024; Sural et al., 2025). For example, INS-7 and INS-35 regulate the NSM neuron to adjust pharyngeal pumping rates (Sural et al., 2025). Additional insulin family members such as INS-3 and INS-11 function in gut-to-brain signaling pathways (Lee and Mylonakis, 2017; Veuthey et al., 2025). Several non-insulin peptides are also involved. FLP-2 (FMRFamide-like peptide) acts via the interneuron AIY to induce responses to oxidative stress within the animal (Jia et al., 2024). NLP-40 (Neuropeptide-like protein) is released to instruct the posterior enteric neurons, AVL and DVB, to elicit steps of the digestive/defecation motor program; its release is tied to a periodic calcium wave that occurs in well-fed animals with a regular frequency of 45-55 seconds (Choi et al., 2023; Mahoney et al., 2008; Wang et al., 2013).
This regular calcium wave may trigger release of additional peptides that influence nervous system behavior more broadly. Several mutants with poor digestive waste release originally identified due to their constipated phenotypes turn out to encode integral components of peptide processing and release such as egl-3, egl-21, aex-1, aex-4, and aex-5 (Husson et al., 2007; Khawand et al., 2026; Mahoney et al., 2008). At least one peptide, NLP-40, is released in time with the digestive motor program, via activation of synaptotagmin-2 (snt-2), a calcium-sensing protein that triggers DCV fusion (Wang et al., 2013). The tyramine response pathway offers additional evidence of calcium-triggered peptide release (Veuthey et al., 2025). This enticing evidence suggests that the rhythmic calcium flux that triggers NLP-40 release, and other steps of the digestive/defecation motor program, may be used more broadly to influence behavioral choices where feeding and nutritional status are relevant. With this notion in mind, we explored an observation of social feeding behavior in a mutant that alters intestinal calcium wave dynamics.
The innexin-16 (inx-16) gap junction subunit is critical for periodic intestinal calcium waves associated with the digestive motor program; loss of function inx-16 mutants disrupt the intestinal calcium wave and greatly reduce the digestive motor program steps that require NLP-40 release (Peters et al., 2007). During routine maintenance of inx-16(tm1589), we observed increased aggregation of well-fed worms and became intrigued. To explore this further, we assayed for social feeding behavior quantitatively. For comparison, we included N2, a solitary strain, and a social feeding strain, npr-1(ad609). The gene, npr-1, encodes a C. elegans receptor related to neuropeptide Y and is neuronally expressed. When active, NPR-1 inhibits the tendency of animals to aggregate in groups, suppresses oxygen level preferences, and affects bordering (de Bono and Bargmann, 1998; Gray et al., 2004). The npr-1 allele we used, ad609, has been characterized to be a strong loss of function allele, equivalent to a genetic null (de Bono and Bargmann, 1998). Our results show that inx-16 mutant animals maintain contact with other animals more frequently than wild-type (N2) solitary worms; young adult inx-16(tm1589) animals demonstrated a significant difference in the number of inx-16 animals found in direct contact with their peers compared to N2 (~0% vs. ~15%; Figure 1A). Yet, the inx-16 phenotype is much weaker than the npr-1(ad609) allele (~80% vs. ~15%; Figure 1A).
To verify that INX-16 functions within the intestine to cause the observed tendency to aggregate, we employed tissue specific rescue of inx-16 mutants. Our and others' inx-16 expression analyses indicated that inx-16 was only expressed in the intestine, yet two studies have provided phenotypic evidence suggesting additional areas of function (Bhattacharya et al., 2019; Liu et al., 2013; Sojka et al., 2025). Intestine-specific rescue was achieved using a construct with a short, well-defined fragment, regulatory sequence of the egg yolk protein, vitellogenin-2, (vit-2) gene, placed upstream of an INX-16 translational fusion (MacMorris et al., 1992). Prior work using this construct led to full rescue of the defecation defects in adults (Peters et al., 2007). Likewise, solitary feeding behavior was restored in the inx-16+pvit-2 intestinal rescue line (Figure 1A). By contrast, driving expression of inx-16 in body wall muscles, using the myo-3 promoter, was unable to restore solitary (wild-type) feeding behavior (Figure 1A; Liu et al., 2013). These data suggest that intestinal INX-16 function contributes to the animal's feeding behaviors.
Having shown that inx-16's actions in the intestine can rescue the social feeding phenotype, we queried whether aggregated feeding was a common feature of mutants with impaired digestive waste release (Thomas, 1990). Defecation mutants induce constipation. One constipated mutant with defects in the expulsion step, exp-1, disrupts an excitatory GABA-ergic receptor in enteric muscles and has been shown to mildly increase social feeding behavior (Beg and Jorgensen, 2003; Bendesky et al., 2012). We assayed this mutant, and others, selecting a set of mutants that affect each defecation motor step and all tissue types involved in the motor program (Branicky and Hekimi, 2006; Thomas, 1990). Two genes required for the first motor step (the posterior body contraction), the intestinal sodium proton exchanger, pbo-4/nhx-7, and the downstream proton-gated ion channel, pbo-5, localized to body wall muscles, were tested (Beg et al., 2008; Pfeiffer et al., 2008). Neither of these Pbo mutants showed increases in social feeding in comparison to N2 (Figure 1B). The later steps of the program, anterior body contraction and enteric muscle contraction, require NLP-40 release from the intestine to stimulate GABA release from enteric neurons, AVL and DVB (Wang et al., 2013). NLP-40 binding activates AEX-2, a g-protein coupled receptor, on these neurons (Allman et al., 2016; Mahoney et al., 2008). Like the Pbo mutants, aex-2(sa3) showed no significant increase over wild-type in our aggregation assay (Figure 1B). In our assays, exp-1(sa6) did not increase aggregation, which differs from prior findings that reported a small but significant increase (Bendesky et al., 2012). Though our defecation mutant panel is not exhaustive, these data suggest that the social feeding phenotype of inx-16 is not broadly shared with mutants suffering from constipation; disrupting defecation per se does not alter social feeding behavior in C. elegans.
Two key gene pathways control the choice between solitary and social feeding behavior: npr-1 and daf-7. Therefore, we used genetic assays to elucidate potential connections between inx-16 and these known pathways (de Bono and Bargmann, 1998; de Bono et al., 2002). Double mutants of inx-16 with daf-7(e1372) and npr-1(ad609) were constructed and tested alongside single mutants to determine whether the double mutant lines had enhanced aggregation. The combination of inx-16 and daf-7(e1372) enhanced aggregation substantially, with the inx-16;daf-7 double mutant exhibiting increased aggregation compared to the daf-7 mutant alone, approximately 23% for the double compared to ~13% for either single mutants (Figure 1C). This additive effect is consistent with inx-16 functioning at least partly independently of daf-7. By contrast, the combination of inx-16 and the strong loss of function npr-1 allele, ad609, did not augment the social feeding level relative to that of npr-1 alone. The inx-16;npr-1 double mutants exhibit no increase in aggregation compared to the npr-1 mutant alone (60% for the double compared to 58% for the single mutant, Figure 1C). The results of a two-way ANOVA analysis support a highly significant genetic interaction between inx-16 and npr-1 (P < 0.003; F = (1, 20)=19). These findings are consistent with inx-16 functioning via the npr-1 pathway to mediate social feeding behavior.
Since our findings placed inx-16 within the npr-1 pathway rather than the daf-7 pathway, we searched for NPR-1 ligands with evidence of intestinal expression. Of the two identified NPR-1 ligands, FLP-18 and FLP-21, flp-21 showed intestinal expression in addition to a subset of neurons (Kubiak et al., 2003; Rogers et al., 2003). The flp-21 mutation causes a significantly increased propensity for animals to aggregate, enhancing aggregation to ~15% (Figure 1D). Interestingly, flp-21's magnitude of impact on feeding behavior is similar to that of inx-16 mutation, ~15% for flp-21 vs. ~14% for inx-16 (Figure 1D; Rogers et al., 2003). Thus, we hypothesized that FLP-21 could be released from the intestine following calcium waves in a manner analogous to NLP-40 release. To begin to investigate this model, we explored epistasis between inx-16 and flp-21. The double mutant line exhibited an aggregation profile very similar to that of either individual mutant strain (inx-16;flp-21 = ~ 13%; Figure 1D). Statistical analysis for gene interaction upholds this epistatic relationship (P < 0.0001; F = (1, 28) = 89). Our results support a model placing FLP-21 release downstream of INX-16 function, and by inference the rhythmic intestinal calcium wave that directs the digestive motor program (Dal Santo et al., 1999; Espelt et al., 2005; Teramoto and Iwasaki, 2006).
Due to flp-21's reported intestinal localization, we suggest that inx-16's social feeding phenotype may be due to reduced FLP-21 release from the intestine. Since NPR-1 signaling dampens social feeding, loss of FLP-21 would increase aggregation, as seen in both inx-16 and flp-21 mutants. Our model is supported by the previously reported epistasis analysis of flp-21(pk1601) and npr-1(ad609); this flp-21 allele did not enhance npr-1(ad609)'s social feeding level, matching our results (Figure 1C; Rogers et al., 2003). Given that flp-18, the other known NPR-1 ligand, is not expressed in the intestine, flp-18 presumably remains active in the inx-16 mutant, allowing ample residual suppression of social feeding to result in the modest aggregation seen in both the inx-16 and flp-21 mutants (Figure 1D; Kapahi et al., 2010).
In summary, our findings demonstrate a behavioral feeding phenotype associated with disrupted intestinal calcium wave dynamics. We propose that this phenotype is due to disruption of FLP-21 mediated activation of NPR-1. Moreover, the social feeding phenotype of inx-16 suggests that the periodic intestinal calcium waves serve to regulate peptide release broadly, influencing a host of behaviors beyond defecation and adding to the growing body of evidence illustrating the importance of gut-to-brain signaling for animal behavior.
","references":[{"reference":"Allman E, Wang Q, Walker RL, Austen M, Peters MA, Nehrke K. 2016. Calcineurin homologous proteins regulate the membrane localization and activity of sodium/proton exchangers in C. elegans. American Journal of Physiology-Cell Physiology 310: C233-C242.
","pubmedId":"","doi":"10.1152/ajpcell.00291.2015"},{"reference":"Artan M, Jeong DE, Lee D, Kim YI, Son HG, Husain Z, et al., Lee. 2016. Food-derived sensory cues modulate longevity via distinct neuroendocrine insulin-like peptides. Genes & Development 30: 1047-1057.
","pubmedId":"","doi":"10.1101/gad.279448.116"},{"reference":"Beg AA, Ernstrom GG, Nix P, Davis MW, Jorgensen EM. 2008. Protons Act as a Transmitter for Muscle Contraction in C. elegans. Cell 132: 149-160.
","pubmedId":"","doi":"10.1016/j.cell.2007.10.058"},{"reference":"Beg AA, Jorgensen EM. 2003. EXP-1 is an excitatory GABA-gated cation channel. Nature Neuroscience 6: 1145-1152.
","pubmedId":"","doi":"10.1038/nn1136"},{"reference":"Bendesky A, Pitts J, Rockman MV, Chen WC, Tan MW, Kruglyak L, Bargmann CI. 2012. Long-Range Regulatory Polymorphisms Affecting a GABA Receptor Constitute a Quantitative Trait Locus (QTL) for Social Behavior in Caenorhabditis elegans. PLoS Genetics 8: e1003157.
","pubmedId":"","doi":"10.1371/journal.pgen.1003157"},{"reference":"Bhattacharya A, Aghayeva U, Berghoff EG, Hobert O. 2019. Plasticity of the Electrical Connectome of C. elegans. Cell 176: 1174-1189.e16.
","pubmedId":"","doi":"10.1016/j.cell.2018.12.024"},{"reference":"BRANICKY R, HEKIMI S. 2006. What keeps C. elegans regular: the genetics of defecation. Trends in Genetics 22: 571-579.
","pubmedId":"","doi":"10.1016/j.tig.2006.08.006"},{"reference":"Choi U, Hu M, Zhang Q, Sieburth D. 2023. The head mesodermal cell couples FMRFamide neuropeptide signaling with rhythmic muscle contraction in C. elegans. Nature Communications 14, 4218 (2023).
","pubmedId":"","doi":"10.1038/s41467-023-39955-8"},{"reference":"Dal Santo P, Logan MA, Chisholm AD, Jorgensen EM. 1999. The Inositol Trisphosphate Receptor Regulates a 50-Second Behavioral Rhythm in C. elegans. Cell 98: 757-767.
","pubmedId":"","doi":"10.1016/s0092-8674(00)81510-x"},{"reference":"de Bono M, Bargmann CI. 1998. Natural Variation in a Neuropeptide Y Receptor Homolog Modifies Social Behavior and Food Response in C. elegans. Cell 94: 679-689.
","pubmedId":"9741632","doi":"10.1016/s0092-8674(00)81609-8"},{"reference":"de Bono M, Tobin DM, Davis MW, Avery L, Bargmann CI. 2002. Social feeding in Caenorhabditis elegans is induced by neurons that detect aversive stimuli. Nature 419: 899-903.
","pubmedId":"","doi":"10.1038/nature01169"},{"reference":"Espelt MV, Estevez AY, Yin X, Strange K. 2005. Oscillatory Ca2+ Signaling in the IsolatedCaenorhabditis elegansIntestine. The Journal of General Physiology 126: 379-392.
","pubmedId":"","doi":" 10.1085/jgp.200509355"},{"reference":"Gray JM, Karow DS, Lu H, Chang AJ, Chang JS, Ellis RE, Marletta MA, Bargmann CI. 2004. Oxygen sensation and social feeding mediated by a C. elegans guanylate cyclase homologue. Nature 430: 317-322.
","pubmedId":"","doi":"10.1038/nature02714"},{"reference":"Husson SJ, Janssen T, Baggerman G, Bogert B, Kahn‐Kirby AH, Ashrafi K, Schoofs L. 2007. Impaired processing of FLP and NLP peptides in carboxypeptidase E (EGL‐21)‐deficient Caenorhabditis elegans as analyzed by mass spectrometry. Journal of Neurochemistry 102: 246-260.
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