Stress induced hyperphagia in C. elegans as a genetic model for binge eating disorders

Summary

Binge eating (BE) is often triggered by stress, dieting, and adverse environmental factors, and represents a common precursor to eating disorders such as anorexia, bulimia, and BE disorder. Stress-induced hyperphagia captures core features of binge-like feeding and shares neurobiological substrates will BE disorder, making it a useful behavioral readout for dissecting underlying mechanisms. We used the genetically tractable worm Caenorhabditis elegans to examine how starvation and stress interact to drive feeding, quantifying pharyngeal pumping as a measure of food intake. Combined starvation and heat stress increased feeding beyond starvation alone, with effects shaped by stress timing, food quality, and sex. Feeding phenotypes were separable from locomotor and feeding-capacity defects. Mutations in evolutionarily conserved homeostatic and hedonic feeding-pathway genes showed distinct hyperphagic responses, implicating that conserved neuronal circuits govern stress-induced feeding across species. This model offers a platform for genetic screening and drug testing to accelerate discovery of therapeutics for binge-like feeding disorders.

Graphical abstract

Highlights

• •Starvation plus heat stress synergistically induce hyperphagia in C. elegans
• •CRF, NPY, and dopamine signaling are essential for stress-induced feeding
• •Hermaphrodites but not males show robust stress-induced hyperphagia
• •Food quality shapes stress-induced feeding more than sensory detection

Behavioral neuroscience; Molecular genetics; Model organism

Introduction

Binge eating (BE) is a complex behavioral phenomenon involving recurrent overeating episodes¹ and constitutes a core feature of multiple eating disorders, including BE disorder (BED), bulimia nervosa, and anorexia nervosa.^2,3 BED is the most prevalent eating disorder, affecting approximately 2.8% of adults—significantly higher than other eating disorders.⁴ Women are disproportionately affected, though this sex disparity is narrowing.⁵ Beyond psychological impacts, BED is associated with serious medical comorbidities including obesity, type 2 diabetes, cardiovascular disease, and metabolic syndrome, creating a substantial burden on global healthcare systems.⁶

BE involves complex interactions between homeostatic and hedonic feeding systems. Homeostatic regulation, mediated by hypothalamic circuits (NPY, AgRP, and POMC neurons), maintains energy balance by monitoring nutritional and metabolic signals.^7,8 Hedonic feeding systems, centered on mesolimbic dopaminergic pathways (ventral tegmental area to nucleus accumbens), mediate food reward and eating motivation.⁹ Stress dysregulates these systems through the hypothalamic-pituitary-adrenal (HPA) axis and corticotropin-releasing factor (CRF) signaling, promoting hyperphagia (overeating) in vulnerable individuals.¹⁰ Stress-induced alterations in dopaminergic signaling enhance palatable food rewards, creating a neurochemical basis for compulsive eating.¹¹

Environmental and psychological factors, particularly stress, frequently precipitate BE episodes.^12,13 Dietary restraint and restrictive eating create vulnerability by establishing a restraint-binge cycle, where food restriction increases the likelihood of subsequent BE episodes,^14,15 especially pronounced in individuals with chronic dieting or weight cycling histories.

Food availability, quality, and palatability are critical determinants of BE behavior. High-fat, high-sugar foods preferentially trigger BE episodes through enhanced reward circuit activation and stress-dampening properties mediated by opioidergic and dopaminergic systems.^16,17 Stress exposure combined with palatable (or attractive) food access increases BE vulnerability through interactions between stress systems and food reward mechanisms.¹⁸ Existing preclinical models provide valuable insights but have significant limitations constraining translational utility. Rodent models, while phylogenetically closer to humans, require extensive time, substantial resources, and present ethical considerations limiting experimental throughput.¹⁹ Mammalian feeding circuit complexity often precludes precise mechanistic dissection of individual pathway contributions. Current rodent paradigms use stress combined with dietary restriction to elicit binge-like eating.^20,21,22 Although these models recapitulate key phenomenological aspects of human BE, they struggle to isolate specific neurobiological mechanisms.^23,24 Additionally, genetic manipulation in rodents remains technically challenging and time-consuming, limiting high-throughput mechanistic studies and drug screening applications. The worm C. elegans offers unique advantages for studying feeding behaviors and their dysregulation. Its nervous system is completely mapped with fully characterized synaptic connectivity.^25,26,27 Despite its apparent simplicity, C. elegans exhibits sophisticated feeding behaviors mediated by evolutionarily conserved molecular pathways showing remarkable homology to mammalian systems.²⁸ Approximately 40% of C. elegans genes have direct human orthologs based on extensive sequence conservation, providing a robust foundation for translational studies.²⁹ The genetic amenability of C. elegans represents perhaps its greatest experimental advantage, as do the organism’s short generation time (3–4 days), its transparent body facilitating live imaging, and its outstanding genetic toolkit that enables the rapid and straightforward generation, manipulation, and analysis of mutants targeting specific genes and pathways of interest.³⁰ The availability of extensive mutant collections facilitates systematic dissection of individual pathway contributions to complex behaviors.

C. elegans feeding is mediated by the pharynx, a neuromuscular pump that ingests bacterial food through rhythmic contractions measurable as pharyngeal pumping rate (PPM).^31,32,33 C. elegans discriminates between bacterial food sources and exhibits dietary choice behaviors, preferring high-quality bacteria that support optimal growth and development.^34,35 The evolutionarily conserved endocannabinoid system further modulates these hedonic feeding aspects, with anandamide modulating olfactory sensitivity to induce hedonic feeding responses.³⁶ This quantifiable feeding behavior responds to diverse environmental stimuli, including food quality,^35,37 starvation,^28,31 and stress conditions,^38,39 providing an accessible experimental readout for feeding studies.⁴⁰ Olfactory perception of food abundance can modulate dietary restriction responses through brain-to-gut signaling, extending lifespan, and revealing sophisticated sensory-metabolic integration.⁴¹ Critically, molecular pathways governing C. elegans feeding, including neuropeptidergic signaling (NPY/NPR-1), stress responses (CRF/SEB-3), and dopaminergic systems (DAT-1/DOP-2), show strong evolutionary conservation with mammalian counterparts.^42,43,44

Given the urgent need for better BE therapeutic approaches, we sought to establish C. elegans as a genetics-focused model to study stress-induced hyperphagia and binge-like feeding responses, and to systematically dissect the contributions of conserved neurobiological pathways to stress-induced feeding alterations. The primary objectives of our study were: (1) to establish experimental conditions that reliably evoke stress-induced hyperphagia in C. elegans through combined starvation and stress protocols; (2) to validate that the observed feeding alterations represent genuine binge-like feeding responses rather than secondary effects of locomotor or sensory deficits; (3) to investigate the role of food attractiveness in modulating stress-induced feeding responses; (4) to systematically characterize the contributions of evolutionarily conserved neurobiological pathways to stress-induced hyperphagia using targeted genetic approaches; and (5) to examine potential sex differences in stress-induced hyperphagia susceptibility using hermaphrodite and male worms. By achieving these objectives, we aim to establish the worm C. elegans as a powerful new model for understanding the neurobiological mechanisms of stress-induced hyperphagia and binge-like feeding, and thereby to facilitate the discovery of novel therapeutic interventions for eating disorders.

Results

Effects of starvation and heat stress on eating behavior (re-feeding) of C. elegans

Eating behavior, in humans and animals, is influenced by factors such as availability and the nutritional or hedonic value of food, by environmental and internal physiological (including stressful) conditions, and by the genetic disposition of the individual. In humans, one or more of these aspects being problematic can lead to eating disorders (EDs). For example, dieting/starvation in combination with negative environmental stress can lead to BE episodes, particularly in individuals suffering from an ED. Here, we wanted to test whether we could model stress-induced hyperphagia and binge-like feeding responses in the worm C. elegans by exposing worms to starvation and re-feeding in combination with environmental stress.

C. elegans feeding behavior is executed by the pharynx (mouth), a tubular muscle structure that pumps bacterial food from the tip of the mouth into the intestine (Figures 1A and 1B). Pharyngeal pumping consists of coordinated cycles of muscle contractions and relaxation, whereby the movements of the pharyngeal grinder are easily visible and recordable under the microscope (Figure 1B). Populations of C. elegans worms consist nearly exclusively of hermaphrodites (effectively females with a sperm-producing organ). Males represent less than 0.5% of a given population. Unless specifically stated otherwise, all experiments were carried out with hermaphrodite worms.

Figure 1.

C. elegans feeding anatomy and stress-induced changes in pharyngeal pumping

(A) A schematic depiction of the alimentary system in the worm C. elegans. The pharynx, the feeding organ (mouth), is in the head of the worm (green highlight in the microscopy image). The intestine extends along the entire length of the worm’s body, constituting the primary site of digestion and nutrient absorption, and terminates in the hindgut, which connects to the rectum and anus through which undigested material is excreted (from Wormatlas; www.wormatlas.org).

(B) C. elegans feeds by pumping and grinding bacterial food through the pharynx into the intestine (not shown). Pharyngeal pumping occurs by contraction and relaxation of radially oriented pharyngeal muscles, which causes the grinder to move back and forth. The pharyngeal grinder is a high-contrast structure, and its movements are easily visible and recordable under the light microscope. Images are reproduced with permission from www.wormatlas.org.

(C) Wild-type C. elegans one-day-old adult animals’ feeding behavior (pharyngeal pumping per minute, PPM) was recorded after different time periods of starvation stress (0, 1, 3, 5, 24 h) at room temperature (23°C) and is presented as mean ± SEM of n = 3 independent experiments, with 5 one-day-old adult animals analyzed per experiment (biological replicates). Statistical differences were determined by one-way ANOVA with Bonferroni multiple comparisons test. ∗p < 0.05 versus 0 h (upper part of C).

(D) Wild-type C. elegans one-day-old adult animals’ feeding behavior (PPM) was recorded after exposure to high-temperature stress (30°C). Worms were exposed to 30°C for different periods of time (15, 30, 45, 60, 90, 180 min) and are presented as mean ± SEM of n = 3 independent experiments, with 5 one-day-old adult animals analyzed per experiment (biological replicates). Statistical differences were determined by one-way ANOVA with Bonferroni multiple comparisons test. ∗p < 0.05 versus 23°C.

First, we examined the response of C. elegans to starvation stress. Wild-type worms were starved for different periods of time (0–24 h) and then re-fed. We measured feeding behavior on food (pharyngeal pumping per minute, PPM) at regular intervals (15, 30, 45, 60, 90, 120, 150, 180 min) (Figure 1C). Prior exposure to starvation induced statistically significant differences in feeding rates (PPM) between starvation groups, whereby the increase in PPM was highest when worms were starved for 3 h as compared to non-starved animals (0 h), while excessive starvation (24 h) proved to be deleterious and led to strongly decreased feeding behavior.

We then separately examined the response of wild-type worms to an environmental stress factor, exposure to high temperature (30°C), which also induced changes in feeding behavior (Figure 1D). PPM was significantly increased in worms under heat stress for up to 60 min compared with non-stressed worms. Again, excessive heat stress (180 min) led to a significant decrease in feeding behavior. We have also examined feeding behavior under more extreme heat stress at 37°C and cold stress at 14°C. Both regimens lead to excessive lethality, and therefore, determination of statistically relevant PPM was not possible (data not shown).

We conclude that exposing C. elegans worms to (moderate) starvation and heat stress induces increased feeding behavior as quantified by increased pharyngeal pumping rates (PPM).

Combining starvation and heat stress induces stress-induced hyperphagia in C. elegans

In humans, dieting/starvation in combination with negative environmental stress is a frequent trigger and key etiological determinant of BE behavior, which includes hyperphagia. To investigate whether a comparable stress-feeding interaction could be observed in C. elegans, we applied starvation stress in combination with heat stress following the experimental framework described above (Figure 1).

We examined feeding behavior (PPM) on food after a defined period of starvation (3 h), combined or not with heat stress at 30°C for different periods of time (Figure 2). We found that PPM, already increased following starvation, was even further increased in worms exposed to heat stress (30°C) as compared to worms kept at room temperature (23°C). This increase in PPM under heat stress strongly correlated with both the timing and different durations of heat stress, whether applied early or late following starvation (Figures 2A–2F). The experimental setup presented in Figure 2B most clearly showed the increase in PPM associated with starvation combined with heat stress and was thus used for all further analyses (Figures 3, 4, 5, and 6).

Figure 2.

Combined starvation and heat stress effects on wild-type feeding behavior

Wild-type C. elegans one-day-old adult animals’ feeding behavior (pharyngeal pumping per minute, PPM) after 3 h of starvation with or without the addition of heat stress (30°C) for different periods of time. Black lines and symbols = animals on starvation stress; blue lines and symbols = animals on food after starvation; red lines and symbols = animals on food after starvation with the addition of heat stress. Data are presented as mean ± SEM of n = 3 independent experiments, with 5 one-day-old adult animals analyzed per experiment (biological replicates). Statistical differences were determined by two-way ANOVA with Tukey’s multiple comparisons test. The experimental setup highlighted (boxed) in (B) was used for all further experiments (Figures 3, 4, 5, and 6). For all (A)–(H) ∗p < 0.05 versus “Starved + Food”.

Figure 3.

Stress-induced hyperphagia across sensory, motor, and physiological mutants

C. elegans one-day-old adult animals’ feeding behavior (pharyngeal pumping per minute, PPM) after 3 h of starvation with or without the addition of heat stress (30°C) for different periods of time (color-coding as in Figure 2): different types of mutants (see Table 1 for details) were tested using the same experimental setup as shown in Figure 2B.

(A) unc-4(e120) mutant worms are movement-impaired and uncoordinated.

(B) eat-2(ad1116) mutant worms feed with an overall reduced feeding speed.

(C) osm-6(p811) mutant worms are defective in their smell and taste modalities.

(D) pbo-4(sa300) mutant worms display a constipated intestine.

(E) hsf-1(sy411) mutant worms display an acquired thermotolerance clearly different from wild type. Data are presented as mean ± SEM of n = 3 independent experiments, with 5 one-day-old adult animals analyzed per experiment (biological replicates). Statistical differences were determined by two-way ANOVA with Tukey’s multiple comparisons test. For all (A–E) ∗p < 0.05 versus “Starved + Food”.

Figure 4.

Food quality modulates stress-induced hyperphagia in wild-type and osm-6 mutants

C. elegans one-day-old adult animals’ feeding behavior (pharyngeal pumping per minute, PPM) after 3 h of starvation with or without the addition of heat stress (30°C) for different periods of time (color-coding as in Figure 2): wild-type worms and osm-6(p811) smell- and taste-defective mutants were fed different types of food and were then tested using the same experimental setup as shown in Figure 2B.

(A and D) Highly attractive (glucose-enriched) food was used.

(B and E) Unattractive (aztreonam-treated) food was used.

(C and F) Food “treated” with DMSO was used as a control. Data are presented as mean ± SEM of n = 3 independent experiments, with 5 one-day-old adult animals analyzed per experiment (biological replicates). Statistical differences were determined by two-way ANOVA with Tukey’s multiple comparisons test. For all (A–F), ∗p < 0.05 versus “Starved + Food”.

Figure 5.

Homeostatic and hedonic pathway mutants alter stress-induced feeding responses

C. elegans one-day-old adult animals’ feeding behavior (pharyngeal pumping per minute, PPM) after 3 h of starvation with or without the addition of heat stress (30°C) for different periods of time (color-coding as in Figure 2): mutants representing homeostatic and hedonic eating/feeding pathways (see Table 1 for details) were tested using the same experimental setup as shown in Figure 2B.

(A and B) Mutants for the C. elegans ortholog of the human neuropeptide Y (NPY) receptor (homeostatic pathway) were used: npr-1(ky13) and npr-1(ad609).

(C and D) Mutants for the C. elegans ortholog of the corticotropin-releasing factor (CRF)/hormone receptor (hedonic pathway) were used: seb-3(eg696gf) and seb-3(tm1848). Note: gf = gain-of-function. Data are presented as mean ± SEM of n = 3 independent experiments, with 5 one-day-old adult animals analyzed per experiment (biological replicates). Statistical differences were determined by two-way ANOVA with Tukey’s multiple comparisons test. For all (A–D) ∗p < 0.05 versus “Starved + Food”.

Figure 6.

Sex-dependent stress-induced hyperphagia

C. elegans one-day-old adult animals’ feeding behavior (pharyngeal pumping per minute, PPM) after 3 h of starvation with or without the addition of heat stress (30°C) for different periods of time (color-coding as in Figure 2): hermaphrodite (A) and male (B) worms were tested in an identical unc-4 mutant background (see STAR Methods) using the same experimental setup as shown in Figure 2B. Data are presented as mean ± SEM of n = 3 independent experiments, with 5 one-day-old adult animals analyzed per experiment (biological replicates). Statistical differences were determined by two-way ANOVA with Tukey’s multiple comparisons test. For both (A and B) ∗p < 0.05 versus “Starved + Food”.

Several EDs are characterized by recurrent episodes of BE. Therefore, we also tested the worm’s ability to react to the repeated application of starvation or heat stress in our feeding assays. Again, upon being starved or heat-stressed twice, worms reacted with significantly increased PPM as compared to the respective control groups (Figures 2G and 2H).

Using our assay system, we were thus able to reproduce and faithfully mimic in worms hyperphagic behavior akin to hyperphagia as part of a human BE episode: starvation combined with stress led the worms “to eat much more within a short period of time”, while in that same time period in worms not exposed to stress, satiety conditions had already set in and feeding behavior had slowed down. Moreover, we could demonstrate in worms’ major features observed in human BE behavior: association with negative environmental stress, correlation with the timing and duration of stress, and recurrence. Thus, we have established a reproducible, quantitative, and versatile experimental setup for studying eating/feeding behaviors, including abnormalities such as hyperphagia as part of BE. Our setup permits easy management of parameters such as the amount or type of food ingested, or variations in the timing and duration of stress.

Genetic underpinnings of stress-induced hyperphagia in C. elegans

Highly human-relevant aspects associated with stress-induced hyperphagia can easily be investigated in C. elegans. Using our experimental setup (cf. Figure 2B) and worm strains carrying defined mutations in genes with direct and strong orthology relationships to the respective human counterparts (Table 1),^{29,45,46,47,48,49,50,51,52} we can clearly separate and distinguish core aspects of stress-induced hyperphagia from associated and potentially confounding features and phenotypes.

Table 1.

C. elegans strains used in this study

Strain name	Genotype	Phenotypes	Molecular change (mutation)	Human ortholog	Reference	Origin
JT5010	wild-type, Bristol N2	reference strain	–	–	–	P Swoboda lab
JT5009	wild-type, Bristol N2	reference strain (male stock)	–	–	–	P Swoboda lab
CB120	unc-4(e120)	impaired movement	splice site	UNC/UNCX homeobox transcription factor	(White, Southgate, and Thomson 1992)45	P Swoboda lab
DA1116	eat-2(ad1116)	reduced feeding speed	splice site	acetylcholine-gated cation- selective channel CHRFAM7A	(McKay et al. 2004)46	P Swoboda lab
PR811	osm-6(p811)	smell and taste defective	splice site	intraflagellar transport 52 (IFT52)	(Collet et al. 1998)47	P Swoboda lab
PS3551	hsf-1(sy441)	acquired thermotolerance	nonsense	heat shock transcription factor (HSF1 and HSF2)	(Hajdu-Cronin, Chen, and Sternberg 2004)48	P Swoboda lab
CX4148	npr-1(ky13)	aggregation behavior on food	nonsense	neuropeptide Y receptor (NPY1R)	(de Bono and Bargmann 1998)38	P Swoboda lab
DA609	npr-1(ad609)	aggregation behavior on food	missense	neuropeptide Y receptor (NPY1R)	(de Bono and Bargmann 1998)38	P Swoboda lab
JT300	pbo-4(sa300)	constipation	missense	potassium:proton antiporter sodium:proton antiporter	(Beg et al. 2008)49	E Jorgensen lab
EG10	pbo-4(ox10)	constipation	frameshift	potassium:proton antiporter sodium:proton antiporter	(Beg et al. 2008)49	E Jorgensen lab
BZ1202	seb-3(eg696gf)	modulates locomotory and arousal states	missense	corticotropin-releasing hormone receptor	(Jee et al. 2013)43	LR Garcia lab
BZ1203	seb-3(tm1848)	modulates locomotory and arousal states	deletion	corticotropin-releasing hormone receptor	(Jee et al. 2013)43	LR Garcia lab
CB904	unc-38(e264)	impaired movement	missense	neuronal acetylcholine receptor subunit alpha-6	(Fleming et al. 1997)50	CGC
RM2702	dat-1(ok157)	dopamine deficient	deletion	sodium-dependent noradrenaline transporter SLC6A2 (solute carrier family 6 member 2)	(Nass et al. 2002)51	CGC
LX702	dop-2(vs105)	dopamine deficient	deletion	dopamine neurotransmitter receptor dopamine receptor D2 (DRD2) dopamine receptor D3 (DRD3)	(Ezcurra et al. 2011)52	CGC

All strains carrying mutations were outcrossed with wild type at least 2–5 times.

All mutations listed are loss-of-function or reduction-of-function mutations, with the exception of seb-3(eg696gf), which is a gain-of-function mutation.

According to standard C. elegans nomenclature, genotype designations are italicised.

CGC: Caenorhabditis Genetics Center, University of Minnesota, St. Paul, MN, USA (https://cgc.umn.edu).

We investigated whether active food searching was necessary for worms to show binge-like feeding responses and found that this was not the case. Two uncoordinated mutants, unc-4 (e120) (Figure 3A) and unc-38 (e264) (Table 2), which present different types of strongly impaired movement phenotypes, both showed binge-like feeding responses very similar to wild-type worms. Using eat-2 (ad1116) (Figure 3B), a mutant with strongly reduced pharyngeal pumping abilities, we could demonstrate that feeding speed as such does not impact stress-induced hyperphagia: worms still showed strongly increased pharyngeal pumping rates (PPM) similar to what was observed in wild-type worms.

Table 2.

C. elegans feeding behavior (pharyngeal pumping per minute/PPM) after 3 h of starvation with or without the addition of heat stress

Genotype	Pharyngeal pumping (PPM)								Stress
	15	30	45	60	90	120	150	180
unc-38(e264)	100.00 ± 1.13	98.14 ± 1.67	99.57 ± 1.63	96.80 ± 1.17	100.76 ± 1.81	100.76 ± 1.81	92.93 ± 1.69	90.18 ± 1.97	No
					104.37 ± 2.18	101.50 ± 1.94	105.12 ± 3.47∗	93.36 ± 3.13	Yes
pbo-4(ox10)	104.92 ± 1.71	109.20 ± 2.99	105.75 ± 2.39	103.08 ± 2.02	106.75 ± 2.73	104.61 ± 1.84	97.49 ± 2.01	99.30 ± 1.84	No
					92.07 ± 8.35	115.60 ± 2.75∗	116.63 ± 3.68∗	110.82 ± 1.75∗	Yes
dop-2(vs105)	100.00 ± 2.27	93.68 ± 1.11	95.52 ± 1.90	96.79 ± 1.63	96.05 ± 1.63	94.65 ± 1.25	96.19 ± 2.89	89.30 ± 2.23	No
					96.84 ± 4.88	92.31 ± 2.46	94.31 ± 2.70	91.88 ± 1.00	Yes
dat-1(ok157)	100.00 ± 0.88	100.88 ± 2.22	102.15 ± 1.86	106.76 ± 2.37	104.50 ± 1.39	99.96 ± 1.98	95.84 ± 1.92	96.58 ± 1.74	No
					100.42 ± 2.06	98.84 ± 2.83	92.88 ± 2.03	91.75 ± 1.42	Yes

Blue numbers = animals on food; red numbers = animals on food with the addition of heat stress; PPM was assessed at the time points indicated in minutes.

Statistics: ∗p < 0.05 versus starved.

Chemosensory modalities such as smell and taste have a strong influence on human eating behaviors. Using the mutant osm-6 (p811) (Figure 3C), with strongly reduced chemosensory capabilities due to defective sensory cilia on all the chemosensory neurons of C. elegans, we again observed wild-type-like stress-induced feeding responses. Similarly, to test whether a state of intestinal fullness and satiety would impact stress-induced hyperphagia, we used two different constipated mutants, pbo-4 (sa300) (Figure 3D) and pbo-4 (ox10) (Table 2). In both cases, BE behavior was unaffected and wild-type-like. We could thus demonstrate that the influence of sensory information highly relevant for eating/feeding behaviors can experimentally be clearly separated from stress-induced hyperphagia.

We have used a regimen of starvation and heat stress to elicit BE behaviors in worms. Therefore, we used a heat-shock factor mutant displaying acquired thermotolerance, hsf-1 (sy441), as a control for our BE assay setup. As expected, these mutants did not react to high-temperature stress, and no changes in pharyngeal pumping rates (PPM) were observed in starved and stressed animals as compared to worms just starved (Figure 3E).

Stress-induced hyperphagia is affected by food quality

Numerous studies in humans and in rodent animal models have highlighted that BE behavior is strongly influenced by the type and quality of food: BE is more likely to occur when palatable or attractive food is available.^53,54 In C. elegans, we examined the differential effects of food quality on stress-induced feeding responses in both wild-type and osm-6 (p811) mutant worms, which have impaired sensory neuron function (Figure 4). All animals underwent a 3-h starvation period prior to experimental feeding conditions and were subsequently exposed to either highly attractive (glucose-enriched) or unattractive (aztreonam-treated) food under both standard and stress conditions. Tukey’s multiple comparisons test revealed that exposure to highly attractive food induced a significant increase in pharyngeal pumping rate in wild-type worms that were both starved and stressed compared to those that were only starved (Figure 4A). By contrast, when stressed worms were exposed to the unattractive food source, their pumping rate did not significantly differ from that of worms that were only starved (Figure 4B), suggesting that food attractiveness is a crucial factor in stress-induced feeding behavior modulation.

When the same experimental conditions were applied to osm-6 mutants, we observed remarkably similar effects, indicating that the chemosensory deficits in these mutants did not eliminate their differential response to food quality under stress. Specifically, glucose supplementation generated a significant increase in the PPM of osm-6 mutants stressed for 30, 60, and 90 min (Figure 4D). Consistent with our observations in wild-type worms, aztreonam-treated food did not elicit increased feeding behavior in stressed osm-6 mutants (Figure 4E).

To rule out potential confounding variables, we conducted control experiments to verify that DMSO, the solvent used to solubilize the antibiotic aztreonam, did not independently affect feeding behavior. Wild-type and osm-6 mutant worms were fed OP50-1 E. coli treated with DMSO (10 μg/mL). As reported in Figures 4C and 4F, respectively, the pharyngeal pumping rates were comparable to those observed in standard conditions for both wild-type (Figure 2B) and osm-6 mutant worms, confirming that DMSO at the concentration used did not significantly impact feeding behavior.

Homeostatic and hedonic aspects of stress-induced hyperphagia in C. elegans

Homeostatic and hedonic mechanisms work in concert to regulate food intake and energy balance in a flexible, adaptive manner that accounts for environmental conditions. To investigate these conserved stress-modulated feeding pathways in our study, we explored these homeostatic and hedonic feeding-associated pathways that are evolutionarily well-conserved in C. elegans, focusing on the NPY-like receptor (NPR-1), the CRF-like receptor (SEB-3), and components of the dopaminergic system (DAT-1 and DOP-2).

The C. elegans gene npr-1 encodes the conserved homeostasis- and appetite-associated Y (NPY) receptor. Using specific mutants commonly employed to genetically dissect natural variation in worm feeding behaviors, we studied feeding behavior under stressful environmental conditions. As shown in Figures 5A and 5B, npr-1 (ky13) and npr-1 (ad609) mutants were not susceptible to environmental variation (starvation and stress), suggesting that NPY signaling is required for adaptive stress-induced feeding responses to changing environmental conditions.

The C. elegans gene seb-3 encodes the conserved CRF receptor (hedonic axis). Available seb-3 mutants, including a gain-of-function allele, seb-3 (eg696gf), and a loss-of-function allele, seb-3 (tm1848), allowed us to address how the intensity of feeding behavior changes under stressful conditions (Figures 5C and 5D). As reported in Figure 5C, animals with the seb-3 gain-of-function allele (eg696gf) were hypersensitive to starvation and stress conditions, exhibiting particularly strong stress-induced hyperphagia. Two-way analysis of variance (ANOVA) with Tukey’s correction revealed a significant increase in pharyngeal pumping movements (PPM) in stressed worms at several observation time points (120, 150, and 180 min). In contrast, the loss-of-function mutant seb-3 (tm1848) did not respond to these conditions (Figure 5D), indicating that SEB-3 activity is necessary for stress-induced hyperphagia.

The dopamine system plays a pivotal role among the most important evolutionarily conserved pathways governing stress-induced feeding behavior in humans. We investigated the roles of the dopamine transporter and dopamine receptor in BE using specific worm mutants: dat-1 (ok157) and dop-2 (vs105) (Table 2). Both strains did not respond to our experimental regimen, suggesting that, also in C. elegans, intact dopaminergic signaling is essential for the expression of stress-induced hyperphagia.

Sex differences in stress-induced hyperphagia—also in C. elegans

Epidemiological studies have consistently demonstrated that EDs exhibit a marked male-female disparity, with anorexia nervosa, bulimia, and BED being significantly more prevalent in females than males.^5,55,56,57 This sexual dimorphism suggests the presence of sex-specific underlying neurobiological mechanisms that influence vulnerability to ED. To explore these mechanisms, we capitalized on well-characterized sexual dimorphisms of the C. elegans nervous system, which encompasses structural, functional, and output differences between hermaphrodites and males.^25,58,59 To maximize comparability, for these experiments we used a genetic crossing scheme involving the unc-4 mutation, which yielded homozygous (unc-4/unc-4) movement-impaired cross-progeny worms, both hermaphrodites and males, coming from the same parent animal (see STAR Methods). The unc-4 mutation does not compromise C. elegans feeding behaviors (cf. Figures 2B and 3A) and thus allows for clear assessment of stress-induced hyperphagia. We compared the behavioral response of unc-4 hermaphrodites and males under combined starvation and stress conditions (exposure to elevated temperature of 30°C for 30 min followed by 2 h of starvation) versus starvation alone (Figure 6). Statistical analysis using Tukey’s multiple comparisons revealed a significant increase in pharyngeal pumping frequency (PPM) in hermaphrodites subjected to stress and starvation compared to the starvation-only group (p < 0.01; Figure 6A). This behavior can be interpreted as stress-induced compensatory hyperphagia, a phenomenon also observed in murine models of EDs⁶⁰ and in human female patients with bulimia nervosa.¹³ In contrast, unc-4 males showed no significant difference in feeding behavior between the two experimental conditions (Figure 6B).

Discussion

Our study establishes a robust and reproducible model of stress-induced hyperphagia in the worm C. elegans that faithfully recapitulates key phenomenological and mechanistic features of binge-like feeding responses while reflecting the specific behavioral architecture of this organism, thereby partially reproducing human binge eating disorder (BED). Our comprehensive experimental approach has yielded several important findings that advance our understanding of the neurobiological mechanisms underlying compulsive feeding behaviors and provide a novel platform for therapeutic discovery. Our primary finding demonstrates that the combination of starvation stress (3 h) with environmental heat stress (30°C) reliably induces significantly increased feeding behavior, as measured by pharyngeal pumping rates (per minute; PPM), compared to either stressor alone. This synergistic effect represents a critical breakthrough, as it mirrors the clinical observation that BE episodes in humans, as well as in animal models, are most commonly triggered by the interaction of dietary restraint (“starvation”) with environmental or psychological stressors rather than by single factors.^21,61,62 The magnitude of this effect was substantial, with combined stress conditions producing PPM rates that were significantly elevated compared to starvation alone across multiple time points, demonstrating the robustness and reproducibility of the behavioral response. This increase in PPM was neither observed under more extreme temperature regimens (14°C and 37°C), nor in the absence of food, indicating that the phenotype reflects a specific feeding-related behavioral adaptation rather than a generalized physiological response to environmental stress. Importantly, our temporal analysis revealed that the timing of stress application critically influences the magnitude of the feeding response. Early stress exposure, applied immediately following the starvation period, produced the most pronounced increases in PPM, while delayed stress application resulted in progressively diminished effects. This time-dependent sensitivity aligns remarkably well with human clinical data showing that acute stress proximal to food exposure represents a particularly potent trigger for BE episodes.¹³ Notably, the observed increase in pharyngeal pumping rate occurs specifically in direct temporal association with stress exposure, rather than as a generalized, context-independent elevation in feeding activity. This stress-contingent nature of the response represents a relevant parallel with the stress-induced triggering of BE episodes in humans, even though the continuous nature of pharyngeal pumping in C. elegans differs fundamentally from the discrete, time-limited structure of BE episodes in mammalian models and in humans.

The finding that stress effectiveness diminishes with temporal distance from the starvation period suggests that there may be a critical window of vulnerability during which stress-feeding interactions are maximally effective, a phenomenon that could have important therapeutic implications.

Our demonstration of behavioral recurrence represents another crucial validation of the model’s clinical relevance. When animals were subjected to repeated cycles of starvation and stress, they continued to exhibit enhanced feeding responses, and importantly, these responses appeared to be maintained or even amplified with repetition. This finding directly parallels clinical and pre-clinical observations that BE episodes tend to cluster and become self-perpetuating over time, with individuals showing increased vulnerability to subsequent episodes following initial BE events.^63,64 The preservation of stress responsiveness across multiple exposures suggests that the underlying neural circuits maintain their sensitivity to environmental triggers, which may explain the chronic, relapsing nature of human EDs. The genetic dissection experiments revealed that stress-induced hyperphagia in C. elegans can be effectively isolated from potentially confounding phenotypes, a finding that represents a significant methodological advance over existing animal models. Our systematic analysis of locomotor-deficient mutants (unc-4 and unc-38) demonstrated that severely impaired movement does not prevent the expression of BE-like feeding responses, indicating that active food searching behavior is not a prerequisite for the observed feeding increases. This finding is particularly important because it establishes that enhanced PPM reflects genuine changes in feeding motivation rather than secondary effects of altered locomotor activity or food accessibility. Similarly, our investigation of sensory-deficient mutants (osm-6) revealed that chemosensory impairments, which dramatically reduce the ability to detect and respond to food-related chemical cues, do not eliminate stress-induced hyperphagia. This result was initially surprising, given the known importance of sensory systems in feeding regulation, but it suggests that the stress-feeding interactions we observed operate through central motivational circuits that can function independently of detailed sensory input about food quality or availability. The preservation of binge-like feeding responses in constipated mutants (pbo-4) further reinforces this interpretation, demonstrating that internal satiety signals related to gut fullness do not override the stress-induced feeding drive. Perhaps most importantly, our analysis of feeding-capacity mutants (eat-2) provided crucial evidence that the observed behavioral changes reflect alterations in feeding motivation rather than mechanical feeding capacity. eat-2 mutants, which have severely reduced pharyngeal pumping capacity due to defects in pharyngeal muscle function, still exhibited proportional increases in PPM under stress conditions despite their baseline feeding limitations. This finding strongly argues that the stress-induced changes observed reflect central alterations in feeding drive that can be expressed regardless of the mechanical constraints on food intake capacity.

Our investigation of food quality effects yielded compelling results that strongly support the translational relevance of the model. When stressed and starved animals were presented with glucose-enriched food (attractive food), they exhibited significantly greater increases in PPM compared to animals receiving standard bacterial food. The magnitude of this effect was substantial, with glucose supplementation producing PPM increases that were approximately 40–50% greater than those observed with standard food under identical stress conditions.⁶⁵ Conversely, when animals were presented with aztreonam-treated bacteria (unattractive food that is difficult to consume), the stress-induced feeding increases were virtually eliminated, with PPM rates remaining at levels comparable to non-stressed controls. These findings regarding attractive food are particularly significant because they directly parallel the clinical phenomenon where human BE episodes preferentially involve highly palatable or attractive, calorie-dense food rather than nutritionally balanced alternatives.^66,67,68 The fact that osm-6 mutants, despite their severe chemosensory deficits, showed similar quality-dependent responses suggests that food attractiveness effects in our C. elegans model operate through mechanisms that extend beyond simple taste or odor detection, possibly involving post-ingestive nutritional sensing or metabolic feedback systems,⁶⁹ or endocannabinoid-mediated modulation of hedonic feeding responses.³⁶

Around 40% of C. elegans genes have direct human orthologs based on extensive primary sequence conservation.²⁹ Capitalizing on this conservation, our systematic analysis of neurobiological pathways provided crucial mechanistic insights into conserved stress-modulated feeding pathways. The mutants examined in this study were selected based on existing literature directly linking their human and murine orthologs to eating disorders and stress-induced feeding dysregulation. Neuropeptide Y signaling (NPR-1 orthology), corticotropin-releasing factor receptor pathways (SEB-3 orthology), and dopaminergic transmission (DAT-1 and DOP-2 orthologies) have all been strongly implicated in binge-like and stress-induced hyperphagia in mammalian models and in human genetic studies. We note, however, that the data presented demonstrate alterations in feeding rate under stress conditions and do not directly resolve the underlying neural circuit architecture at a mammalian complexity level. Accordingly, we frame these findings as evidence for the conservation of stress-modulated feeding pathways, rather than making claims regarding the direct equivalence of BE circuits across species. Notably, the investigation of CRF-like signaling through seb-3 mutants⁴³ yielded particularly striking results. Gain-of-function seb-3 (eg696gf) mutants demonstrated hypersensitivity to the combined starvation and stress protocol, exhibiting dramatically increased PPM rates that were significantly elevated above wild-type responses at multiple time points (120-, 150-, and 180-min post-stress). This hypersensitivity suggests that enhanced CRF signaling amplifies stress-feeding interactions, consistent with the known role of CRF in mediating stress responses and clinical observations that individuals with hyperactive stress response systems are more vulnerable to BE.⁷⁰ Conversely, loss-of-function seb-3 (tm1848) mutants showed complete loss of stress responsiveness, with PPM rates remaining at baseline levels even under the combined starvation and stress conditions that reliably elicited robust responses in wild-type animals. This complete abolition of stress-induced feeding suggests that CRF-like/SEB-3 signaling is not merely modulatory but represents an essential component of the neuronal circuitry mediating stress-feeding interactions.⁷¹ The dramatic phenotypic differences between gain- and loss-of-function alleles provide strong evidence for a dose-dependent relationship between CRF(-like) signaling intensity and stress-induced hyperphagia vulnerability.

The investigation of NPY-like signaling through npr-1 mutants revealed equally important mechanistic insights. Both npr-1 (ky13) and npr-1 (ad609) loss-of-function mutants failed to exhibit normal stress-induced feeding responses, with PPM rates remaining relatively stable across stress conditions, which otherwise elicited consistently robust responses in wild-type controls.^72,73 This finding suggests that NPY-like/NPR-1 signaling plays a critical permissive role in enabling stress-feeding interactions, consistent with the known function of NPY systems in coordinating feeding responses to environmental challenges and energy deficit states.⁷⁴ Moreover, recent evidence demonstrates that sensory perception of food, particularly olfactory cues, can bidirectionally regulate these homeostatic responses through brain-to-gut communication pathways that influence both feeding behavior and longevity.⁴¹The failure of npr-1 mutants to respond to stress conditions is particularly noteworthy because these animals retain normal baseline feeding capabilities and can respond to other environmental stimuli, indicating that the defect is specifically related to stress-feeding integration rather than general feeding or stress response mechanisms.⁴² This specificity supports the hypothesis that NPR-1 represents a critical node in the neuronal circuitry⁷² that translates stress signals into altered feeding motivation.

Our analysis of dopaminergic signaling revealed compelling evidence for the conservation of reward system involvement in stress-modulated feeding pathways. Both dat-1 (ok157) mutants (lacking the dopamine transporter) and dop-2 (vs105) mutants (lacking a key dopamine receptor) showed complete inability to mount stress-induced feeding responses, despite maintaining baseline feeding capabilities.^39,44 The fact that manipulations targeting different components of the dopaminergic system (reuptake and reception) produced similar phenotypes strongly suggests that intact dopaminergic neurotransmission is essential for the expression of binge-like feeding responses.

These dopaminergic findings are particularly significant given the central role of mesolimbic dopamine systems in human BE pathophysiology and the growing body of evidence implicating reward system dysfunction in EDs.^75,76 The conservation of dopaminergic modulation of stress-feeding interactions between C. elegans and mammals suggests that insights gained from the worm model regarding conserved stress-modulated feeding pathways may have direct translational relevance for understanding and eventually treating human EDs.⁷⁷ Our demonstration of sex-specific differences in stress-induced hyperphagia susceptibility provides additional important validation for the clinical relevance of the C. elegans model. Hermaphrodites showed robust stress-induced feeding responses, with significant PPM increases. In contrast, males showed minimal, if any, responses to the same stress conditions, with PPM rates remaining largely unchanged from baseline levels. This sex difference is particularly compelling because it mirrors the well-established female predominance for human ED and suggests that the neurobiological mechanisms underlying this sex bias may be evolutionarily ancient and amenable to study even in simpler model systems.^4,55

The sex-specific differences we observed likely reflect the documented sexual dimorphism in the C. elegans nervous system, including sex-specific neurons and circuits involved in feeding and stress responses.^25,59 These sex-specific differences in behavioral response are possibly mediated by the AVP and DVA neurons, which exhibit sexual dimorphism and are involved in modulating food intake in response to stress in C. elegans.⁷⁸ Furthermore, gene expression analyses have revealed that C. elegans genes involved in serotonin and dopamine signaling, key neurotransmitters also implicated in human EDs,^79,80 are differentially regulated in the two sexes in response to stress.^78,81 Our findings strengthen the hypothesis that sex-specific differences in neuronal circuits regulating feeding and stress response contribute to the differential vulnerability to EDs. The evolutionary conservation of these mechanisms from worm to human suggests that C. elegans represents a valid model for studying the neurobiological bases of these sex-specific differences in stress-induced hyperphagia, with potential implications for the development of male-versus female-targeted therapies. The primary strength of our C. elegans stress-induced hyperphagia model lies in its unparalleled genetic amenability, which enables the systematic dissection of individual pathway contributions to a complex behavioral phenotype. Unlike mammalian models, where genetic manipulations are time-consuming and challenging, C. elegans allows rapid generation and analysis of targeted mutants, facilitating comprehensive mechanistic studies within practical time frames. Our demonstration that stress-induced hyperphagia can be isolated from locomotor, sensory, and feeding capacity phenotypes represents a significant methodological advance that allows precise attribution of behavioral changes to specific neurobiological mechanisms, separate from potential secondary effects of genetic manipulations. Such mechanistic precision is extremely difficult to achieve in mammalian systems due to the complex interconnectivity of feeding circuits and, in many instances, the pleiotropy of most genetic manipulations.

The C. elegans model offers substantial advantages in terms of experimental efficiency and reproducibility compared to existing approaches. The short generation time (3–4 days), small size, and minimal maintenance requirements enable high-throughput studies with large sample sizes, improving statistical power and reproducibility. The standardized environmental conditions and genetically identical populations eliminate many sources of variability that plague mammalian studies, including genetic background effects, housing conditions, and stress handling. The quantitative nature of the pharyngeal pumping readout (PPM) provides objective, reproducible measurements of feeding behavior that are less subject to observer bias than many behavioral assessments used in mammalian models. This quantitative precision, combined with the ability to perform repeated measurements on the same animals over time, enables detection of subtle behavioral changes and detailed characterization of temporal dynamics.

Limitations of the study

Several limitations should be acknowledged when interpreting the translational relevance of our findings in C. elegans. A fundamental constraint relates to the organizational differences between worm and mammalian feeding behavior. Human feeding is typically organized into discrete meals separated by periods of non-eating, with complex regulatory mechanisms governing meal initiation, duration, and termination. In contrast, C. elegans feeding represents a much more continuous process driven by rhythmic pharyngeal pumping when food is available, lacking the clear temporal structure that characterizes mammalian feeding patterns. While the stress-contingent increase in PPM we describe occurs in direct temporal association with the experimental stressor, continuous pharyngeal pumping cannot fully capture the episodic, time-limited character of BE episodes in humans or in established mammalian models. This represents an inherent limitation of the behavioral readout that should be considered when interpreting the model’s translational scope. Indeed, increased pharyngeal pumping following stress and deprivation captures only quantitative hyperphagia, lacking the subjective and emotional dimensions central to the diagnosis of human BE.¹ Therefore, our model explores stress-induced hyperphagia with compulsive-like characteristics rather than a complete recapitulation of the human disorder characterized by cognitive components (loss of control, guilt, distress) and social contexts.

The nervous system architecture differs substantially between species. C. elegans possesses 302 neurons compared to >86 billion in humans and lacks discrete brain structures, including regions critically implicated in mammalian feeding regulation.^7,9 While entire neurotransmitter systems are evolutionarily conserved, their circuit-level organization differs markedly. Nevertheless, the conservation of fundamental signaling mechanisms, such as stress-responsive neuropeptides, monoaminergic modulation, food-reward associations, and brain-to-gut communication pathways,⁴¹ provides substrate for identifying core molecular pathways therapeutically targetable across species. C. elegans lacks several physiological systems contributing to mammalian eating behavior. The gut-brain axis, including bi-directional communication between the gastrointestinal tract, gut microbiota, and the central nervous system, represents a noteworthy difference.⁸² Emerging evidence demonstrates that gut-brain axis dysregulation contributes to eating disorder pathophysiology through altered microbial metabolites, intestinal permeability changes, and vagal signaling.⁶⁴ C. elegans, while possessing a simple digestive tract, cannot adequately model the complex immunological, endocrine, and neuroanatomical features of the mammalian gut-brain axis. Additionally, the absence of cortisol/corticosterone signaling and complex hypothalamic-pituitary-adrenal axis feedback mechanisms limit stress-response modeling. Furthermore, sex-specific aspects of stress-induced hyperphagia cannot entirely be addressed in C. elegans, as sex-specific disparities in human BE very likely also include mammalian-specific factors such as sex hormones, psychosocial influences, and cultural factors.⁶²

We note the absence of pharmacological validation in our current study. We have not yet demonstrated that the stress-induced hyperphagia phenotype can be modulated by compounds known to affect feeding or binge-like behavior in mammalian systems. While the genetic amenability of C. elegans and the quantitative nature of the PPM readout make it theoretically well-suited for high-throughput compound screening, the translational relevance of such an approach will require demonstration that pharmacological agents targeting conserved feeding-related pathways, such as NPY, CRF, or dopaminergic signaling, produce the expected modulatory effects on stress-induced hyperphagia in the worm. Future studies should prioritize such a pharmacological validation as a critical step toward establishing the worm model as a translational drug discovery platform for binge-like feeding disorders.

Despite its apparent simplicity, the C. elegans model demonstrates promising translational relevance through the conservation of key neurobiological pathways and behavioral features. The systematic validation of pathway conservation, combined with the phenomenological parallels described above, suggests that therapeutic targets identified in this model may have direct relevance for human EDs. This potential will need to be confirmed through pharmacological studies demonstrating that compounds with known efficacy in mammalian models of binge-like feeding can produce comparable effects in the worm paradigm. The model’s amenability to high-throughput drug screening⁸³ represents a potentially valuable feature for therapeutic development, as the small size, short lifespan, and simple maintenance requirements of C. elegans enable cost-effective screening of large compound libraries, while the quantitative behavioral readout (PPM) facilitates automated analysis approaches. These features could contribute to accelerating the identification of novel therapeutic candidates for binge-like feeding disorders, which currently lack specific pharmacological treatments.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Mariangela Pucci (mpucci@unite.it).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •All data reported in this article will be shared by the lead contact upon request.
• •This article does not report original code.
• •Any additional information required to re-analyze the data reported in this article is available from the lead contact upon request.

Acknowledgments

In addition to KI institutional funding, PS was also supported by the following grant providers: Swiss Research Foundation (Vetenskapsrådet), Swedish Brain Foundation (Hjärnfonden), Åhlén Foundation. Work by MP in the laboratory of PS was supported by exchange grants from the EU COST Action 1408 and by grants from the Bo and Vera Ax:son Johnsson Foundation and the Henning and Johan Throne-Holst Foundation. This work was supported by the Italian Ministry of University and Research under grants FISR2019_00202 -DIE TAMI to C.D.

Author contributions

M.P.: conceptualization, data curation, formal analysis, investigation, methodology, project administration, writing – original draft, and writing – review and editing; C.C.: writing – review and editing; C.D.: conceptualization, funding acquisition, supervision, and writing – review and editing; P.S.: conceptualization, data curation, methodology, funding acquisition, project administration, resources, supervision, writing – original draft, and writing – review and editing.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains
E. coli OP-50–1	P Swoboda lab	–
Chemicals, peptides, and recombinant proteins
Aztreonam	Sigma-Aldrich	PZ0038
D-(+)-glucose	Sigma-Aldrich	G8270
Experimental models: organisms/strains
Wild type, Bristol N2	P Swoboda lab	JT5010
Wild type, Bristol N2 (male stock)	P Swoboda lab	JT5009
unc-4(e120)	P Swoboda lab	CB120
eat-2(ad1116)	P Swoboda lab	DA1116
osm-6(p811)	P Swoboda lab	PR811
hsf-1(sy441)	P Swoboda lab	PS3551
npr-1(ky13)	P Swoboda lab	CX4148
npr-1(ad609)	P Swoboda lab	DA609
pbo-4(sa300)	E Jorgensen lab	JT300
pbo-4(ox10)	E Jorgensen lab	EG10
seb-3(eg696gf)	LR Garcia lab	BZ1202
seb-3(tm1848)	LR Garcia lab	BZ1203
unc-38(e264)	CGC	CB904
dat-1(ok157)	CGC	RM2702
dop-2(vs105)	CGC	LX702
Software and algorithms
GraphPad Prism10	GraphPad Software	C D’Addario Lab

Experimental model and study participant details

C. elegans strains and maintenance

Worms were propagated at 20°C in 60 mm diameter plates with nematode growth medium (NGM) seeded with E. coli OP50-1 as bacterial food.³⁰ The wild-type Bristol N2 strain (JT5010, hermaphrodites; JT5009, males) was used as the reference strain for all mutants analyzed. All strains used in this study are listed in Table 1, the key resources table.

Method details

Types of food

Standard bacterial food (OP50-1)

E. coli OP50-1 bacteria were obtained from the Caenorhabditis Genetics Center (CGC, University of Minnesota, St. Paul, MN, USA; https://cgc.umn.edu). A single bacterial colony was inoculated in Luria broth (LB) with 50 μg/mL streptomycin and grown overnight at 37°C in a shaking liquid culture. This culture was then concentrated 5-fold by centrifugation at 4000 RPM. Before seeding on to NGM plates containing 50 μg/mL streptomycin, bacteria were diluted 1:1 with LB. For seeding 350 μL of this bacterial suspension were spread on to 60 mm NGM plates and allowed to dry completely.

Attractive bacterial food (OP50-1 on glucose-enriched NGM plates)

To enhance food attractiveness, glucose-enriched NGM plates were prepared as previously described.⁶⁵ Supplementation of the bacterial lawn with glucose has been shown to increase pharyngeal pumping rate and promote fat accumulation in C. elegans, thereby recapitulating key features of dietary-induced hyperphagia and representing a well-established positive control condition for increased food intake.^84,46 In brief, 60 mm NGM plates were entirely covered with 400 μL of glucose (D-(+)-glucose; Sigma Aldrich) from a 1 M stock solution prepared in ddH2O, to reach a desired final concentration of ∼40 mM. These plates were then allowed to dry for 24 h. The next day 350 μL of E. coli OP50-1 were added on top of the glucose layer to cover the entire plates. The bacterial suspension was allowed to dry completely before animals were placed on plates. All plates were used for experimentation within a 7-day period.

Unattractive bacterial food (OP50-1 treated with aztreonam)

The antibiotic aztreonam (Sigma) prevents cellular division and produces filamentous bacteria that worms cannot easily feed on. E. coli OP50-1 treated with aztreonam were prepared following a protocol by Gruninger et al.⁸⁵ Briefly, OP50-1 cultures were grown overnight at 37°C in LB with rigorous shaking. Once the bacterial culture reached logarithmic growth, aztreonam was added to a final concentration of 10 g/mL. The bacterial culture was then grown for an additional 4 h with minimal shaking to minimize damage to the bacterial filaments. For seeding 350 μL of this bacterial suspension were spread on to 60 mm NGM plates and allowed to dry completely before animals were placed on plates. All plates were used for experimentation within a 7-day period.

Measuring pharyngeal pumping rate

Pharyngeal pumping was recorded at room temperature (23°C) in one-day-old adult animals on food: we observed and counted grinder movements in the terminal bulb of the pharynx, whereby a full contraction and relaxation of the radially orientated muscles of the terminal bulb of the pharynx were defined as one complete pharyngeal pumping event (Figures 1A and 1B).⁸⁶ Each data point for a given time interval was obtained from five distinct worms. For each worm, the pharyngeal pumping rate (expressed in pumps per minute, PPM) was measured as the average of three consecutive 10-s observations. The resulting average for each worm was then used as the unit of analysis. Each experiment was performed in triplicate, yielding a total of 15 measurements per worm per condition per time point across the three independent experimental replicates. Each experiment was repeated 2–3 times (total worms 10–15). We used a stereomicroscope to count pharyngeal pumping (Olympus SZX 12; 1.6× PF). To allow comparison of feeding responses across different experimental conditions on a common scale, raw PPM values were converted to arbitrary units (A.U.): the mean PPM value measured at 15 min after the end of the starvation period was set to 100, and all remaining values were normalized proportionally with respect to this reference point. Raw PPM values, not converted to arbitrary units, for all conditions and time points are reported in Table S1.

Starvation stress: Food restriction

Well-fed one-day-old adult worms were transferred to empty NGM plates without food to then be starved for different time periods (ranging from 0 to 24 h). After starvation, worms were transferred back to fresh NGM plates with food and after a 15-min recovery period feeding behavior (pharyngeal pumping) was observed at defined time points during a 180-min time period.

Heat stress: Change of temperature

Sets of worms, previously starved or not, were transferred to fresh NGM plates with food and then exposed to a stressful temperature regimen (30°C) for different time periods (1, 2 or 3 h). The consequences of heat stress on feeding behavior were recorded by measuring pharyngeal pumping as described above.

General experimental outline

Well-fed one-day-old adult worms, maintained at 20°C on NGM plates with food, were acclimated for at least 15 min at room temperature (23°C). After this “settling-in” time period, worms were transferred to empty NGM plates without food to be starved for 3 h at 23°C. After starvation worms were immediately transferred to fresh NGM plates with food and after 15 min of adjustment, their feeding behavior (pharyngeal pumping) was recorded every 15 min for 1 h as described above. Then, the set of worms under investigation was split into two groups and transferred, respectively, to two different NGM plates with food, one to be kept at room temperature (23°C) and the other to be exposed to heat stress (30°C). Pharyngeal pumping rates for both sets of worms (exposed to 23°C and 30°C, respectively) were recorded every 30 min for 2 h. Worms on NGM plates with food that were recovered from the heat stress condition at 30°C were kept at 23°C for 1 min and then pharyngeal pumping was immediately recorded. All NGM plates, with or without food, used for starvation stress, for heat stress and for recording pharyngeal pumping, were always pre-conditioned for at least for 1 h at the required temperature regimen, 23°C or 30°C, as required for the specific experimental setup.

Genetic construction of uncoordinated (unc-4) mutant hermaphrodite and male worms

The C. elegans unc-4 (e120) mutation is a recessive loss-of-function mutation (Table 1) that locates on chromosome II. Homozygous (unc-4/unc-4) mutant hermaphrodites were mated with wild-type (+/+) males. Heterozygous (unc-4/+) male F1 progeny from this first cross were mated in a second cross with homozygous (unc-4/unc-4) mutant hermaphrodites. The F2 progeny from this second cross segregated both hermaphrodites and males that were severely movement impaired and uncoordinated (homozygous unc-4/unc-4), a phenotype that can easily be discriminated from heterozygous worms (unc-4/+), which display wild-type movement phenotypes. The desired F2 cross progeny, unc-4/unc-4 homozygous hermaphrodites and males, was selected two days after egg-hatching and then subjected to comparative feeding behavior assessment as one-day-old adults as described above.

Quantification and statistical analysis

Statistical analyses and graphs were carried out and produced using GraphPad Prism 10 for Windows (GraphPad Software Inc, La Jolla, CA, USA). Statistical differences in pharyngeal pumping rates (PPM) were determined by two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. C. elegans food intake variation after starvation (Figure 1C) was analyzed by one-way analysis of variance (ANOVA), and post-hoc comparisons were made with the Bonferroni multiple comparisons test. Data presented for all assays are the average of n = 3 independent biological replicates, with 5 one-day-old adult animals analyzed per experiment per condition, unless otherwise stated in the corresponding figure legend. All data are expressed as mean ± SEM. p values <0.05 were accepted as being statistically significant. Statistical details of each experiment, including the specific test used, the exact value of n, and the definition of center and dispersion measures, are reported in the corresponding figure legends.