THE NEUROPEPTIDE NEUROMEDIN U RECEPTOR NMUR-1 BUFFERS INSULIN RECEPTOR SIGNALING IN BACTERIA-DEPENDENT C. ELEGANS SURVIVAL

Distinct microbial environments exert diverse e ffects on the physiology and survival of the nematode Caenorhabditis elegans. Here, we show that C. elegans grown on two Escherichia coli strains exhibit different survival dynamics. Wild-type C. elegans on the B type OP50 exhibit more early deaths compared to C. elegans on K-12 type CS180. These early deaths on OP50 are characterized by swollen pharynges (P-deaths) due to bacterial accumulation within the tissue. In contrast, animals on CS180 are more resistant to P -deaths. Th ese bacteria-dependent differences in P-deaths depend on bacterial lipopolysaccharide structures and the activities of the C. elegans neuropeptide neuromedin U receptor nmur-1, which reduces P-deaths on OP50, but not on CS180. Surprisingly , however, nmur-1 promotes the opposite response when the insulin receptor DAF-2 has decreased activity — where nmur-1 now stimulates P-deaths on OP50, but again with no effect on CS180. We also find that nmur-1 acts in sensory neurons to promote its bi-directional effects on longevity, which depend on the FOXO transcription factor daf-16. nmur-1 regulates the expression of the insulin-like peptide daf-28, which further suggests a regulatory mechanism that maintains insulin receptor DAF-2 signaling at a suitable level. Thus, our studies reveal that nmur-1 serves to buffer the dynamic range of DAF-2 signaling, thereby optimizing pharyngeal health and survival in response to specific bacteria. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 3

Bacteria are the major dietary source for some animals [1, 2], provide metabolites as part of the intestinal microbiome [3-5], or act as pathogens in other situations [3, 6]. As these three major bacterial functions impact and shape an animal’s life history traits , animals must adapt and respond to their microbial environment for optimal health and survival. In the nematode worm C. elegans, bacteria serve as its primary food source [1], supplying nutrients that stimulate differential gene expression to affect physiology and survival [7-13]. Since C. elegans eat different kinds of bacteria, its genetic tractability and the ease of studying its physiology have also made it a useful model for microbiome-derived metabolite studies [5, 14]. At the same time, several bacteria have been shown to pose a threat to C. elegans [6]. To isolate the contributions of these bacterial functions, C. elegans physiology can be dissected when grown on specific bacterial types — such as wild-type versus mutant bacteria or live versus dead bacteria or different bacterial species [5-13]. These studies reveal that some bacteria are a source of nutrients, metabolites, and/or infection [10, 11, 13, 15]. In the laboratory, C. elegans usually feeds on a diet of the B-type E. coli OP50 [16], which is not part of the animal’s native microbiome [5]. E. coli OP50 has also been shown to be pathogenic to the worm [15, 17]. Colonization of C. elegans pharynges by live, proliferating OP50 leads to swelling of the pharynx, ultimately killing the animals — a type of death known as P-death [15, 17]. Presently, the mechanism(s) underlying this type of death remain unclear. For example, the OP50 bacteria-derived cue(s) that promote P -deaths are unknown. Previously, we have shown that worms grown on E. coli OP50 live shorter than worms grown on a K-12 type of E. coli, CS180, and that this lifespan difference is at least partly dependent on the E. coli lipopolysaccharide (LPS) structure [7]. Here we show that LPS structure also mediates OP50- dependent P-deaths in C. elegans. In the host, only a few C. elegans genes have been implicated in modulating P-deaths [17-19], which include regulators of innate immunity. Of particular interest is the neuropeptide .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 4 neuromedin U receptor nmur-1, which elicits distinct responses to different pathogenic bacteria [20]. nmur-1 promotes survival against the pathogen Enterococcus faecalis , limits survival on Salmonella enterica, and has no effect on Pseudomonas aeuruginosa [20]. Interestingly, nmur-1, which is expressed in several sensory neurons and in subsets of interneurons and motor neurons [7, 21], has also been shown to mediate the E. coli OP50 -dependent effects on mitochondrial function and longevity in C. elegans [7, 11]. However, the nmur-1 deletion allele, ok1387, used in these studies is also tightly linked to a second mutation, ot611, which is located in the gene filamin-2 (fln-2), whose gene product promotes P-deaths [18]. To dissect the effects of nmur-1 on OP50-dependent P-deaths, we recombined fln-2(ot611) away from nmur-1(ok1387). Here we show that nmur-1 has complex effects on OP50-dependent C. elegans survival but has little or no effect on CS180-dependent worm survival. In the presence of wild-type insulin receptor DAF-2 signaling, the other known regulator of P-deaths [17, 19], wild-type nmur-1 inhibits P-deaths on OP50. Intriguingly, nmur-1 produces an opposite response on OP50 when DAF-2 insulin receptor activity is reduced. In this context, wild-type nmur-1 now increases P-deaths, as well as deaths that are not associated with swollen pharynges (non-P deaths) . This interaction with daf-2 suggests that nmur-1, which acts in sensory neurons, adjusts the dynamic range of insulin receptor signaling, a pathway known to be important for survival (reviewed by [22]). We further find that wild-type nmur-1 specifically regulates the expression of an insulin-like peptide (ILP) ligand, daf-28, suggesting that nmur-1 tunes insulin receptor signaling through defined ILPs. Thus, this mechanism should provide the physiological flexibility necessary in coping with diverse microbial environments.

E. coli LPS structure modulates C. elegans survival dynamics The E. coli B-type OP50 caused worms to live shorter than a K -12 type E. coli, CS180 (Fig 1A; Table 1) [7]. Worms on OP50 also had a higher rate of early deaths compared to worms on CS180 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 5 (Fig 1A), which suggests the presence in OP50 of an early hazard that is absent from CS180. Early deaths on OP50 (P-deaths) due to bacterial colonization can be visualized by swollen pharynges (compare Fig 1D to 1F; [15, 17]). Since CS180 reduced deaths in early adulthood (Fig 1A; Table 1), we tested if P -deaths contributed to the lifespan differences between wild-type worms on the two bacteria (see Materials and Methods on determination of P-deaths ; Fig S1). First, we observed that all P-deaths on OP50 or CS180 occurred by day 15 of adulthood (Fig S2). Second, OP50-fed worms showed about 3 times more P-deaths, when compared to CS180-fed worms (Fig 1B and 1C; Table 1), revealing that worms on CS180 were more resistant to P-deaths. Next, we asked what bacterial cues might contribute to the P-death differences between OP50-grown and CS180-grown worms. We previously showed that the E. coli LPS structure can modulate C. elegans longevity [7]. CS180 LPS truncation mutants, CS2198 and CS2429 (Fig 1G), have been shown to shorten wild-type worm lifespan [7]. Hence, we compared the number of P- deaths on both CS2198 and CS2429 to those on CS180. While we found that only the short LPS mutant E. coli CS2198 decreased worm lifespan in this study (Fig 1H; Table 1), both E. coli strains with the shorter LPS produced more P-deaths than CS180 (Fig 1I and 1J; Table 1). Thus, our

show that altering the core LPS structure is sufficient to promote E. coli colonization of the pharynx and increased P-deaths. Opposing effects of nmur-1 on E. coli OP50 depends on the daf-2 insulin receptor We then asked what host genetic factors influence the bacterial-dependent P -deaths. One candidate gene is the neuropeptide neuromedin U receptor nmur -1, which has been demonstrated to mediate bacteria- specific innate immune responses [20], some of which might depend on the LPS structure of E. coli [7]. The nmur-1(ok1387) deletion mutation used in these studies is tightly linked to the ot611 mutation present in the putative actin-binding scaffold protein gene fln-2, which also regulates P-deaths [18]. To address OP50-dependent P-death phenotypes that are specific to nmur-1, we separated the fln-2(ot611) and nmur-1(ok1387) mutations. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 6 This approach enabled us to dissect the complex effects of the isolated nmur-1(ok1387) mutation. First, nmur-1(ok1387) produced a short lifespan on OP50 but not CS180, whereas the fln-2(ot611) mutant lived long only on OP50 (Fig 2A; Table 1). While fln -2(ot611) mutants also had fewer P-deaths (Fig 2B and 2C; Table 1), the nmur-1(ok1387) mutant had more P-deaths on OP50 but not on CS180 (Fig 2D and 2E; Table 1). These results were recapitulated in a second independent deletion allele of nmur -1, lst1672 [21] (Fig 2F and 2G; Table 1). L oss of nmur -1 affected survival largely through pharynx-dependent deaths (Fig S3A to S3B; Table S1). When we only counted deaths that are characterized by unswollen pharynges (non-P deaths; Fig S3C; Table S1), the survival of nmur-1 mutants is more similar to wild-type survival. In this context, nmur-1 acts to protect C. elegans from P-deaths in a bacterial-dependent manner. Intriguingly, the genetic background alter s the effect of nmur-1 mutations on OP50- dependent deaths. The wild-type insulin receptor DAF -2 promotes deaths caused by bacterial colonization and pharyngeal swelling [17, 19]. In insects and mammals, neuromedin U signaling influences insulin signaling by suppressing insulin secretion under certain contexts [23-27]. Thus, we tested whether the P -death phenotype of nmur-1 is daf-2-dependent. Unexpectedly, the daf-2(e1368) reduction-of-function mutation not only lengthened lifespan and suppressed P- deaths but also revealed that nmur-1 activity has bi-directional effects on lifespan and P-deaths. Unlike animals with wild-type daf-2 (Fig. 2A and 2D to 2G ; Table 1), nmur-1(ok1387) and nmur-1(lst1672) now led to fewer P -deaths in the daf-2 mutant backgrounds ( e1368 or mu150) on OP50 (Fig 3; Table 1), but not on CS180 (Fig 3A; Table 1). Thus, deletion of nmur-1 further extends the long lifespan of daf-2 mutants in a bacteria-dependent manner (Fig 3; Table 1). In contrast to animal s with wild -type daf-2, nmur-1 modulated both P-deaths and non-P deaths when daf-2 activity was reduced (Figs S3 D to S 3F, S 4A and S4C ; Table S1). This suggests that wild-type nmur-1 affects survival through other mechanisms besides pharyngeal colonization. Importantly, the opposing nmur-1 phenotypes in the wild-type versus daf -2 mutant backgrounds suggest that wild-type nmur-1 adjusts and buffers insulin receptor activity. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 7 Specifically, loss of nmur -1 enhances the impact of daf-2 activity on lifespan: it increases the difference in the median time of death between wild type and daf-2 reduction-of-function mutants, when considering either total deaths or P-deaths (Fig 3F). This increase in the dynamic range of median lifespan between wild-type and daf-2 mutants implies that loss of nmur -1 leads to an animal’s greater sensitivity to DAF-2 activity levels. Together, these results suggest a role for wild- type nmur-1 in buffering the impact of DAF-2 receptor activity on lifespan. nmur-1 promotes opposing effects on survival by acting in sensory neurons in response to LPS structure To address the mechanisms through which nmur-1 exerts its multiple activities, we first verified its role through rescue experiments. Expression of wild-type nmur-1 from its own promoter [21] rescued the nmur-1 short-lived single mutant phenotype (Fig 4A; Table 1). When daf-2 activity is reduced, extrachromosomal expression of wild-type nmur-1 from its own promoter also rescued the longer life phenotype due to the nmur-1 mutation (Fig 4C; Table 1) . The same construct rescued both the P-death (Fig 4C; Table 1) and non-P death phenotypes caused by nmur-1(ok1387) in the daf-2(e1368) background (Fig S4A and S4B; Table S1). Next, we sought to determine where nmur-1 acts to influence C. elegans survival. Expression of wild-type nmur-1 from the sensory neuron-specific promoter osm -6p [21] likewise rescued the nmur-1 survival phenotypes in both wild-type and mutant daf-2 backgrounds (Fig 4B and 4D; Table 1). This result suggests that wild-type nmur-1 in sensory neurons inhibits P-deaths when daf-2 activity is high but promotes P deaths under low daf-2 activity. Interestingly, this construct also rescued the non-P death phenotype (Fig S4C and S4D; Table S1) more robustly than the P-death phenotype (Fig 4D; Table 1). Together these data suggest that nmur -1 acts in sensory neurons to exert its multiple, context-dependent effects on C. elegans survival. We also wanted to test whether LPS structure influences nmur-1 activity to modulate P- deaths. E. coli CS180 has little effect on the nmur-1 phenotype in wild-type or mutant daf-2 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 8

(Fig 5A; Table 1). However, a truncation of the LPS structure in E. coli CS2429 recapitulated the nmur-1 phenotypes on OP50 : (i) an increase in P -deaths and shortening of lifespan when daf-2 activity is wild type; and (ii) a decrease in P-deaths and lengthening of lifespan when daf-2 activity is reduced through a mutation (Fig 5B; Table 1). Thus, these findings suggest that LPS structure plays a role in the nmur -1 modulation of infection-dependent P-deaths that is mediated by daf-2 activity. nmur-1 acts upstream of the insulin pathway and regulates the sensory expression of an insulin-like peptide The complex interactions between nmur-1 and the daf-2 insulin receptor motivated us to determine if they act in the same pathway. The FOXO transcription factor daf -16 is the downstream effector of many daf-2 functions that include longevity (reviewed by [22]), leading us to test if nmur-1 phenotypes are also daf-16-dependent. Consistent with prior work [19], we found that loss of daf-16 increased the number of all deaths, including P-deaths (Fig 6A ; Table 1), and suppressed the effect of daf-2 on all types of deaths (Fig 6B; Table 1). All nmur-1-dependent types of deaths also required daf-16 (Fig 6A and 6B; Table 1), suggesting that wild-type nmur-1 modulates these deaths by acting through the DAF-2/DAF-16 signaling pathway. To test whether nmur-1 acts upstream of this pathway, we next determined if nmur -1 affects the expression of some ILPs. C. elegans has forty ILPs that are organized into an ILP-to- ILP network, where some ILPs have been proposed to act as agonists or antagonists of DAF -2 [28]. We focused on two ILPs , ins-6 and daf-28, which are potential DAF -2 ligands with known roles in lifespan and whose expression are modulated by bacteria-derived cues [29-32]. Because ins-6 and daf-28 overlap in expression [29, 32, 33] with nmur-1 in the sensory neuron ASJ [34, 35], we compared the expression of the two ILPs in the ASJ neurons of both control animals versus nmur-1 loss-of-function mutants. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 9 While nmur-1 had no effect on ins -6 expression in ASJ (Fig 6C ), loss of nmur -1 significantly increased daf-28 expression in the same neurons (Fig 6D). Thus, it is possible that nmur-1 alters the expression of ILPs such as daf-28 to exert its effects on survival.

Bacteria can modulate insulin signaling as pathogens, food, or part of the microbiome ([36]; reviewed by [37, 38]), thereby influencing key physiological processes that are important for survival. Here we used C. elegans genetics to dissect the contributions of these interactions on lifespan. By stratifying early and late deaths due to different bacteria-host interactions in a population, we reveal how nmur-1 contributes to the overall survival dynamics under normal and reduced insulin signaling. Through systematic analyses of gene-gene and gene-environment interactions, our findings reveal a new role for the neuromedin U pathway in buffering the effects of the perturbations to insulin signaling during bacteria-host interactions. Neuromedin U receptor NMUR-1 modulates survival dynamics The survival curve of C. elegans is produced primarily by early deaths due to bacterial accumulation in the pharynx that are analogous to infection [15, 17] and late deaths due to other causes. Here we implicate the neuropeptide neuromedin U receptor NMUR-1 as a modulator of both early and late deaths by acting from sensory neurons . While the NMUR-1 effects on early deaths are consistent with a role in pathogen-specific innate immune responses, its effects on late deaths suggest additional role(s). We also show that the NMUR-1 effect on early deaths occurs in response to the bacterial LPS structure. Furthermore, NMUR-1 can exert opposing effects on lifespan depending on DAF-2 receptor activity. More importantly, our findings on the bi -directional effects of NMUR -1 on pharyngeal-dependent survival suggest a model where NMUR-1, which acts in sensory neurons, .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 10 adjusts the dynamic range of insulin receptor signaling to promote tissue health and longevity (Fig 7). Bacterial LPS structure interacts with nmur-1 to influence early death We show that the bacterial LPS structure determines the frequency of early deaths caused by bacterial colonization of the pharynx (Figs 1E, 1G to 1J and 5; Table 1). The LPS might affect C. elegans pharyngeal integrity by changing pharyngeal pumping rates [7]. However, this possibility is not supported by our previous findings that wild-type animals have similar pumping rates on E. coli OP50, CS180 or CS2429, which have different LPS structures ([7]; and references therein). Alternatively, LPS structure might affect bacterial adherence to the pharyngeal tissues, where LPS acts as an important stimulator for the host immune system [39-42]. Some E. coli strains have an O-antigen that promotes adherence to tissues, an important step in pathogenesis [43]; but the strains used here lack an O-antigen (Fig 1G; [7]]. Unlike the O-antigen, the bacterial core LPS has been shown to be less adhesive, although the core LPS may regulate the expression of adherence proteins [44-46]. For example, the truncated LPS core of E. coli CS2198, CS2429 and OP50 might stimulate or hinder specific immune responses in C. elegans. Here we find that LPS structure modulates the two activities of the C. elegans neuropeptide receptor NMUR-1 in altering pharynx-dependent deaths (Fig 5; Table 1). Sun and colleagues have recently shown that nmur-1 regulates different immune responses to specific bacterial pathogens [20]. Wild-type nmur-1 promotes resistance to Enterococcus faecalis, inhibits resistance to Salmonella enterica and has no effect on survival on Pseudomonas aeruginosa [20]. While bacterial LPS has not been directly implicated in these differing responses, all three bacteria have different cell wall and LPS compositions : E. faecalis is a Gram -positive bacterium, which likely lacks an LPS, similar to many Gram -positive bacteria [47, 48], whereas S. enterica and P. aeruginosa are both Gram-negative bacteria with different LPS structures [49, 50]. Interestingly, .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 11 the intestinal accumulation of E. faecalis in nmur-1 mutants [20] is reminiscent of the bacterial colonization of pharynges on the truncated LPS mutant CS2429 (Fig. 5; Table 1). In rodents , LPS -induced responses are also modulated by the neuromedin U (NMU) peptide, a ligand of mammalian NMUR [51-53]. LPS exposure increases the production of the inflammatory cytokine interleukin IL -6 from peritoneal macrophages, which is abolished by the loss of the NMU peptide [52]. In this study [52], the presence of NMU promotes inflammation and LPS-induced mortality. However, in another study [53], NMU is shown to be protective against LPS-induced neuronal death, where NMU promotes the production of the neuroprotective brain- derived neurotrophic factor, BDNF, but has no effect on interleukins . While it is unclear whether the two studies used the same LPS isolate [52, 53], the NMU/NMUR signaling pathway has differing responses to LPS in both C. elegans and rodents. Since the NMU signaling pathway mediates LPS responses in mammals, and LPS has also been shown to affect mammalian insulin activity [54, 55], we propose that the differing nmur-1 responses in C. elegans depend on the levels of insulin receptor activity (Fig 7), as we discuss below. nmur-1 buffers insulin receptor signaling levels to maintain health The insulin signaling pathway regulates C. elegans immune responses (reviewed by [56]), pharyngeal health, and survival [17, 19]. Severe reduction or hyperactivation of insulin receptor activity is deleterious to the animal. Insulin receptor daf -2 null mutants exhibit lethality or embryonic and larval arrest [57], whereas a gain-of-function mutation in daf-2 results in short- lived animals that are vulnerable to stressors [58, 59]. These studies suggest the importance of maintaining insulin receptor signaling levels at an optimal level. Modulators provide a mechanism for fine-tuning insulin receptor activity in fluctuating environments [60]. nmur-1, which is co-expressed with daf-2 insulin receptor and/or its ILP ligands in neurons [7, 21, 34, 61], can serve as a potential modulator of DAF-2 activity in regulating pharyngeal health (Fig 7). Here we show that wild-type NMUR-1 promotes healthy pharynges and prevents death .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 12 (Fig 2), presumably by lessening DAF-2 activity (Fig 7, condition 1). In contrast, when DAF-2 activity is reduced, NMUR-1 inhibits healthy pharynges and increases deaths (Fig 3), likely by upregulating DAF-2 activity (Fig 7, condition 2). These bi -directional effects of NMUR -1 on pharyngeal health and survival are features of neuromodulators, which ensure that cells and tissues signal within an optimal range to function appropriately across different environments [60, 62]. Here we propose that NMUR-1 modulates tissue and cell activities by buffering and preventing large fluctuations in DAF-2 signaling activity. This is supported by how NMUR-1 limits the differences in median survival between wild type and daf-2 reduction-of-function mutants (Fig 3F). NMU signaling suppresses insulin secretion from Drosophila insulin-producing cells [23] and mammalian pancreatic β-cells in some [24-27] but not all contexts [63, 64]. In C. elegans, NMUR-1 may ensure that the insulin receptor signals appropriately by regulating the expression of specific ILP ligands. Consistent with this idea, we show that nmur-1 specifically downregulates the expression of the ILP daf-28, but not of ins-6 (Fig 6C and 6D). At the same time, it is possible that some DAF-2/DAF- 16 targets signal back to NMUR -1 and/or its ligands. Through such a feedback mechanism, NMUR-1 can buffer and modulate the levels of insulin receptor signaling. Thus, in the presence of NMUR -1, the C. elegans insulin receptor is neither hyperactive nor hypoactive in response to the bacteria in the animal’s environment (Fig 7). This model highlights a mechanism that ultimately prevents large deviations in insulin pathway activity (Fig 7), which is necessary in optimizing pharyngeal health and survival. The C. elegans pharynx also resembles the mammalian heart both structurally and mechanistically [65], whose health is susceptible not only to diet [66] but also to bacterial infections [67-69]. Moreover, mammalian insulin signaling plays a role in promoting cardiac health versus disease states [70-72]. Because of the high degree of conservation between C. elegans and mammals, we speculate that the NMUR-1-mediated buffering of insulin receptor signaling in C. elegans might also exist in higher animals. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 13

C. elegans strains and growth conditions All C. elegans mutants used in this study were backcrossed at least three times to wild type. Mutants that were used in the survival assays are reported in Table s 1 and S1 with their genotypes. All experiments were carried out at 25°C. However, all worms were grown for at least two generations at 20oC on the specified bacteria, before they were shifted to 25oC past the dauer larval arrest decision stage, including animals carrying the daf-2(e1368) or daf-2(mu150) mutation [57, 73, 74]. The temperature 20 oC is permissive for growth for daf-2 mutants, which prevents dauer entry, whereas 25°C is non-permissive for these animals [57]. Bacterial strains and growth conditions The bacterial strains that were used in the study are E. coli OP50, E. coli CS180, E. coli CS2198, and E. coli CS2429 (see [7]; and references therein). Bacterial strains were grown from single colonies in Luria-Bertani media at 37°C until the log-phase, with an optical density (OD) of ~0.6 at 600 nm . For the experimental assays , 6-cm Nematode-Growth (NG) agar plates [16] were seeded with approximately 250 μl of bacteria and streaked to cover the entire plate (full-lawn bacterial plates). We used full-lawn plates during the lifespan assays and ILP imaging to prevent the confounding factor of worms avoiding the bacterial lawns [75]. Plates were incubated at 25°C overnight before they were used for any experiment. Recombining nmur-1(ok1387) away from fln-2(ot611) To recombine nmur-1(ok1387) away from fln-2(ot611), which is about 420 kilobases away on chromosome X of the QZ58 C. elegans strain, QZ58 was crossed to wild type. Among the subsequent progeny of the nmur-1 fln-2/+ + cross-progeny, we identified 2 recombination events out of 206 chromosomes: one progeny was homozygous for the fln- 2(ot611) mutation and .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 14 heterozygous for nmur-1(ok1387); another animal was homozygous for nmur-1(ok1387), but not for fln-2(ot611). These animals were allowed to reproduce to isolate the nmur-1 single mutant and the fln-2 single mutant. The mutations were detected by PCR. The ok1387 deletion was detected by using the primers: ok1387 fw (5’-ATA AGT GTC ATA GAT ACA GG-3’); ok1387 rv (5’-AAT ACA TAT ACT GAT TGA CC-3’); and ok1387 int rv (5’- AAT GCT ATG GCA GAG AAG TG -3’). The mutant was detected as a 441-bp band, whereas wild type was detected as a 602-bp band. The ot611 point mutation was detected by using a forward primer whose 3’ end is complementary to the adenine point mutation and generates a 253-bp band with the ot611 reverse primer, 5’-CCT GTC ACA TGA GCA CTA ATG TC-3’. The wild-type allele of fln-2 was detected by using a forward primer whose 3’ end is complementary to cytosine and generates a 253-bp band with the ot611 reverse primer. The presence or absence of the wild-type and ot611 alleles were further confirmed by s equencing. We used the ot611_F primer, 5’-GTC ACT ATA ATA GAC GCC GTA ATG C-3’, and the ot611 reverse primer to generate a 536-bp fragment that was sequenced to determine whether position 301 of the fragment is a C or an A. Lifespan assays Worms were picked for all experiments at the late L4 stage at 25 oC and were transferred onto full lawns of the specified bacteria daily for the first 6 days of adulthood, thereby preventing the mixing of subjects with their progeny. The details of the censoring during experiments are explained in the legend of Table 1. Kaplan-Meier estimates were done using the JMP 8.0.1 software (SAS). P values of both Wilcoxon and l og-rank tests are reported in the data tables. The Wilcoxon test is the better measure of statistic al significance when hazard ratios are not constant throughout an assay [7, 76], which is the case for most of our survival comparisons. Necropsy analysis to determine P-deaths versus non-P deaths .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 15 The pharynges of all the dead animals in survival assays were imaged using a Nikon Eclipse Ni- U microscope and a Photometrics Coolsnap ES2 camera at 400x magnification. The surface area of the terminal pharyngeal bulb (see Fig 1C to 1E) was measured using the NIS-Elements software (Nikon Instruments, Inc). The surface area of the terminal bulb was then divided by the diameter of the body of the same animal at the region of the terminal bulb, which is also known as the grinder (area P/diameterG). This normalization addressed the possibility that the general size of the animals affected the pharyngeal surface area. Through a principal component analysis of dead wild-type animals on OP50 (n = 387) from 8 independent survival assays, we initially separated these animals into two clusters —one with swollen pharynges (P -deaths) and one without swollen pharynges (non-P deaths). Since P - deaths happen early in the lifespan of the population [15], we used areaP/diameterG and the age of death as variables. The principal component analysis was carried out in the R 4. 4.2 software [77], where we plotted the data (Fig S1) using ggplot2 [78] and ggfortify [79]. From Fig S 1, we determined the threshold areaP/diameterG that would separate the two clusters, which was a ratio value of 27. This threshold value was then used to categorize animals that died with significant pharyngeal swelling (P-deaths) or with no pharyngeal swelling (non-P deaths) in all experiments. Imaging ILP: :mCherry expression Generation of the ILP::mCherry transcriptional reporter. The generation of the ins-6p::mCherry reporter drcSi68 is as previously described [33]. The daf-28p::mCherry drcSi98 was generated by flanking the mCherry gene with 3.3-kb sequences upstream of the daf-28 start codon and 4.7- kb sequences downstream of the daf-28 stop codon. Both 5’ and 3’ cis regulatory sequences of daf-28 were amplified from YAC Y116F11 with Phusion DNA polymerase and then cloned into the pCR-Blunt vector, which was sequenced for confirmation. The subsequent reporter was next cloned into a MosSCI vector for integration (pQL184) at the ttTi4348 site of chromosome I. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 16 Live imaging of worms. Animals were grown on full lawns of OP50 at 20 oC, before they were shifted to 25 oC at the second larval stage (L2). Worms were then imaged at 1000x magnification, once they reached the mid -L4 stage at 25 oC, using a Nikon Eclipse Ni -U microscope and a Photometrics Coolsnap ES2 cooled digital camera. We quantified fluorescence intensities using a built-in fluorescence quantification algorithm (NIS -Elements, Nikon Instruments, Inc). The Student’s t-test was used to compare each ILP expression between wild type and nmur-1(ok1387) expression. Statistical analyses Statistical analyses were performed using JMP 8.0.1 (SAS) for all survival assays; GraphPad Prism 8 software for the ILP imaging measurements; and R 4.4.2 for the principal component analyses of the swollen pharynx-dependent deaths. For more details, refer to above and the figure and table legends.

We thank the Caenorhabditis Genetics Center (funded by NIH P40 OD010440), I. Beets, C. Kenyon and J. Watteyne for strains used in this study. We also thank A. Caballero, D. A. Fernandes de Abreu and C. Liu for technical support in generating the drcSi98 strain and M. Friedrich for comments on the manuscript. This work was supported by an ERC Starting Investigator Grant (NeuroAge 242666) and a Research Councils UK Fellowship to Q. C., and by the Novartis Research Foundation, Swiss National Science Foundation (31003A_134958), Wayne State University, the Alcedo family and NIH (R01 GM108962) to J. A. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 17

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The R Journal 8: 474–85. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 26 Figure legends Fig 1. Bacterial food type influences swollen pharynx-dependent deaths in C. elegans. (A) Wild-type C. elegans fed E. coli OP50 had more early deaths compared to C. elegans fed E. coli CS180. (B) The early deaths depended on swollen pharynges (P -deaths) in worms fed OP50 compared to worms fed CS180. (C) The fraction of P-deaths out of 144 total deaths on OP50 or 164 total deaths on CS180. (D) DIC image of a non-swollen pharynx in a live one-day old adult worm. (E-F) DIC images of dead five-day old adult worms that either have a swollen pharynx (E) or a non-swollen pharynx (F). Scale bar is 10 µ m. (G) LPS structures of OP50 and CS180. The red scissors indicate the LPS truncations that correspond to CS2198 and CS2429, which are derived from CS180. (H-I) All deaths (H) versus P-deaths (I) of wild-type C. elegans on CS180 and the LPS-truncated CS2198 and CS2429. (J) The fraction of P -deaths out of 64 deaths on CS180, 61 deaths on CS2429 and 65 deaths on CS2198. Chi -square analyses were carried out to determine significant differences between the fractions of P-deaths among the different groups of animals on the different bacteria in this figure and subsequent figures. *** denotes P < 0.001, whereas **** denotes P < 0.0001. See Table 1 for the rest of the statistical analyses that also pertain to this figure and subsequent figures. Fig 2. Wild-type nmur-1 decreases pharynx-dependent deaths in a bacteria-dependent manner. (A) On OP50, the nmur-1(ok1387) single mutant shortened lifespan whereas the fln-2(ot611) single mutant extended lifespan. On CS180, nmur-1 mutants lived slightly longer than wild type, but fln-2 mutants lived like wild type. (B) fln-2(ot611) had fewer P-deaths on OP50. (C) The fraction of P-deaths on OP50 out of 138 deaths for wild type and 162 deaths for fln-2(ot611). (D) nmur-1(ok1387) had more P-deaths on OP50 but not on CS180. (E) The fraction of P-deaths on OP50 out of 203 deaths for wild type and 231 deaths for nmur-1(ok1387). (F) A second allele of nmur-1, lst1672, also shortened lifespan and increased P deaths on OP50. (G) The fraction of .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 27 P-deaths on OP50 out of 257 deaths for wild type and 269 deaths for nmur -1(lst1672). The following symbols denote the following: **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. Fig 3. In contrast, under low daf-2 insulin receptor activity, wild-type nmur-1 increases P- deaths. (A) In the daf-2(e1368) reduction-of-function mutant background, nmur -1(ok1387) had an opposing effect on lifespan on OP50—nmur -1(ok1387) increased longevity by decreasing P- deaths. Again, nmur-1(ok1387) had little effect on CS180. (B) A second allele of nmur-1, lst1672, also increased lifespan and reduced P -deaths in the daf -2(e1368) mutant background. (C) nmur-1(ok1387) also enhanced the lifespan of another reduction-of -function allele of daf-2 , mu150, which led to fewer P-deaths. (D) The fraction of P-deaths on OP50 out of 203 deaths for wild type, 231 deaths for nmur -1(ok1387) single mutants, 182 deaths for daf-2(e1368) single mutants and 206 deaths for daf-2(31368); nmur-1(ok1387) double mutants out of 2 trials. (E) The fraction of P -deaths on OP50 out of 165 deaths for wild type, 160 deaths for nmur -1(lst1672) single mutants, 105 deaths for daf-2(e1368) single mutants and 156 deaths for daf-2(31368) ; nmur-1(lst1672) double mutants out of 2 trials. (F) Left panel: The difference in the median time of all deaths between wild type and daf-2 reduction-of-function mutants in the presence (n = 6 trials) or absence of nmur -1 (n = 6 trials ). Right panel: The difference in the median time of P- deaths between wild type and daf-2 reduction-of-function mutants in the presence (n = 9 trials) or absence of nmur-1 (n = 10 trials). Significance in median differences is determined by the Mann- Whitney test. The following symbols denote the following: *, P < 0.05; **, P < 0.01. Fig 4. nmur -1 acts in sensory neurons to modulate longevity and P deaths. (A-B) The longevity and P-death phenotypes of nmur-1(ok1387) single mutants that were rescued in nmur- 1-expressing cells (A) or in sensory neurons alone (B). (C-D) The longevity and P -death phenotypes of daf-2(e1368) ; nmur-1(ok1387) double mutants, where nmur -1 was rescued in nmur-1-expressing cells (C) or in sensory neurons alone (D). .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 28 Fig 5. The effects of nmur-1 depend on the bacterial LPS structure. (A-B) The longevity and P-death phenotypes of nmur-1(ok1387) single mutants and daf-2(e1368); nmur-1(ok1387) double mutants on CS180 (A) versus CS2429 (B). Fig 6. nm ur -1 acts upstream of DAF-2 and modulates the expression of the insulin-like peptide daf-28. (A-B) The daf-16-dependence of the longevity and P-death phenotypes of nmur- 1(ok1387) single mutants (A) and of daf-2(e1368) ; nmur-1(ok1387) double mutants (B). (C-D) The effects of nmur-1 on the ASJ neuron expression of the ins -6p::mCherry transcriptional reporter drcSi68 [C; n = 44, wild type; n = 41 , nmur-1(ok1387)] and the daf-28p::mCherry transcriptional reporter drcSi98 [D; n = 45, wild type; n = 44, nmur-1(ok1387)] at mid-L4 on OP50. ** indicates P value < 0.01. Fig. 7. A model for how nmur-1 adjusts DAF-2 receptor activity in regulating survival. See text for details. Table legend Table 1. Cumulative statistics of all deaths versus P-deaths on different bacteria. Worms were censored at the time they crawled off the plate, exploded, or bagged, allowing these worms to be incorporated into the data set until the censor date. This avoids loss of information. P values that are significant (P < 0.05) are italicized and in bold face. If the test population lived longer or had fewer P-deaths than the population to which it is compared, the P values are also underlined. Both Wilcoxon and logrank P values are shown for comparison (see Materials and

on the suitability of one statistical test versus the other). All survival assays were carried out at 25 oC on full lawns of the specified bacteria. The superscripts indicate the population to which the test population is compared. The following abbreviations or symbols indicate: WT, wild .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 29 type; OP, OP50; CS, CS180; ok1387*, the genotype daf-2(e1368); nmur-1(ok1387); $, same data used in Figs 2D and 3A; $$, some of the data were also used in Fig 3A; and CS24, CS2429. Supporting information Fig S1. Principal component analysis (PCA) for necropsy analysis. PCA analysis performed on areaP/diameterG and age of death (days) values of 387 wild-type animals from 8 separate experiments on OP50. Numerical values of datapoints represent the areaP/diameterG of each dead animal. Animals are separated into two clusters—probability ellipses shown in red (left side) and in blue (right side). The red cluster represents animals with swollen pharynges at death (P - deaths). The blue cluster represents animals with non-swollen pharynges at death (non-P deaths). Fig S2. Dist ribution of the total P-deaths over the course of wild-type lifespan on different bacteria. (A-B) P-deaths were no longer observed after day 15 of adulthood on OP50 (A) and on CS180 (B). The number of P deaths on OP50 was 395 out of 1070 deaths (number of trials, 10). The number of P deaths on CS180 was 55 out of 528 deaths (number of trials, 6). Fig S3. nmur-1 has multiple and complex effects on OP50-dependent deaths. (A-C) The survival curves of wild type and nmur-1(ok1387) single mutants (cumulative of 7 independent trials from Figs 2, 3 and 6), when all types of deaths (A) or only P-deaths (B) are included or when P-deaths are excluded (C). (D-F) The survival curves of daf-2(e1368) single mutants and daf-2(e1368); nmur-1(ok1387) double mutants (cumulative of 6 independent trials from Figs 3 and 6), when all types of deaths (D) or only P -deaths (E) are included or when P-deaths are excluded (F). See Table S1 for the statistical analyses of these data. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 30 Fig S4. nmur-1 acts from sensory neurons to modulate non-P deaths when daf-2 activity is reduced. (A-D) The non-P death phenotypes of daf -2(e1368) single mutants versus daf-2(e1368); nmur-1(ok1387) double mutants (A , C), where nmur -1 was rescued in nmur -1- expressing cells (B) or in sensory neurons alone (D). See Table S1 for the statistical analyses of these data. Table S1. nmur -1-dependent P-deaths versus non-P deaths on E. coli OP50. Cumulative statistics of all types of deaths of the indicated C. elegans strains. P values that are significant (P < 0.05) are italicized and in bold face. If the test population lived longer or had fewer P -deaths than the population to which it is compared, the P values are also underlined. The superscripts indicate the population to which the test population is compared. ok1387* indicates the genotype daf-2(e1368); nmur-1(ok1387). The symbol ** denotes that the pharyngeal sizes of late deaths were left unmeasured. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint Sifoglu et al., Figure 1 D E F J Lipid AHepHep Hep Kdo Kdo Kdo inner core NAG Hep Glc Gal Glc Glc outer core Glc Glc Lipid A HepHep Hep Kdo Kdo Kdo inner coreouter core CS2198 CS2429 LPS of E. coli K-12 LPS of E. coli B CS180 OP50G 0 5 10 15 0 255 10 15 20 wild type P - deaths only 0.0 1.0 0.2 0.4 0.6 0.8 fraction alive H wild type age (days of adulthood) CS180 CS2429 CS2198 0.0 1.0 0.2 0.4 0.6 0.8 fraction alive I 0.0 1.0 0.2 0.4 0.6 0.8 fraction of P-deaths CS180CS2429CS2198 0.0 1.0 0.2 0.4 0.6 0.8 fraction of P-deaths C OP50 CS180 0 255 10 15 20 0.0 1.0 0.2 0.4 0.6 0.8 wild type fraction alive age (days of adulthood) A B 0 5 10 15 wild type P - deaths only CS180 OP50 CS180 OP50 **** *** **** .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint A 0.0 1.0 0.2 0.4 0.6 0.8 fraction alive 0 255 10 15 20 age (days of adulthood) 30 35 40 fln-2(ot611)nmur-1(ok1387) wild type OP50 0 255 10 15 20 30 35 40 CS180 B OP50 CS180 0 5 10 15 P - deaths only P - deaths only 0 5 10 15 0.0 1.0 0.2 0.4 0.6 0.8 fraction alive wild type fln-2(ot611) Sifoglu et al., Figure 2 age (days of adulthood) D CS180 0 5 10 15 P - deaths onlyOP50 P - deaths only 0 5 10 15 wild type nmur-1(ok1387) 0.0 1.0 0.2 0.4 0.6 0.8 fraction alive age (days of adulthood) OP50 P - deaths only 0 5 10 150 255 10 15 20 OP50 F 0.0 1.0 0.2 0.4 0.6 0.8 fraction alive wild type nmur-1(lst1672) age (days of adulthood) fraction of P-deaths C wild type fln-2 (ot611) 0.0 1.0 0.2 0.4 0.6 0.8 **** OP50 fraction of P-deaths E wild typenmur-1 (ok1387) 0.0 1.0 0.2 0.4 0.6 0.8 OP50 ** fraction of P-deaths G wild typenmur-1 (lst1672) 0.0 1.0 0.2 0.4 0.6 0.8 OP50 *** .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint age (days of adulthood) OP50 CS180 A 0 255 10 15 20 30 35 40 P - deaths only 0 5 10 15 CS180 P - deaths only Sifoglu et al., Figure 3 B 0 255 10 15 20 30 35 40 0 5 10 15 daf-2(e1368) daf-2(e1368); nmur-1(ok1387)nmur-1(ok1387)wild type fraction alive 0.0 1.0 0.2 0.4 0.6 0.8 nmur-1(lst1672) wild type daf-2(e1368) daf-2(e1368); nmur-1(lst1672) OP50 P - deaths only fraction alive 0.0 1.0 0.2 0.4 0.6 0.8 age (days of adulthood) 0 255 10 15 20 30 35 40 0 5 10 15 OP50 OP50 OP50 P - deaths onlyOP50 nmur-1(ok1387) wild type daf-2(mu150) daf-2(mu150); nmur-1(ok1387) 0 255 10 15 20 30 35 40 0 5 10 15 C fraction of P-deaths 0.0 1.0 0.2 0.4 0.6 0.8 wild type nmur-1(ok1387)daf-2(e1368)daf-2(e1368); nmur-1(ok1387) D ** OP50 OP50 wild type nmur-1(lst1672)daf-2(e1368)daf-2(e1368); nmur-1(lst1672) F 0 4 8 12 16 all deaths difference in median deaths (days) + _ ** P-deaths nmur-1+ _ ** ** * ** E .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 1550 255 10 15 20 0.0 1.0 0.2 0.4 0.6 0.8 fraction alive nmur-1(ok1387); nmur-1p::nmur-1A nmur-1(ok1387)wild type OP50 0 10 P - deaths onlyOP50 age (days of adulthood) 0 255 10 15 20 nmur-1(ok1387); osm-6p::nmur-1B OP50 P - deaths onlyOP50 Sifoglu et al., Figure 4 1550 10 0.0 1.0 0.2 0.4 0.6 0.8 C OP50 P - deaths onlyOP50 0.0 1.0 0.2 0.4 0.6 0.8 fraction alive 0 255 10 15 20 30 35 40 OP50 P - deaths onlyOP50 0 5 10 15 daf-2(e1368); nmur-1(ok1387); nmur-1p::nmur-1 daf-2(e1368)wild type daf-2(e1368); nmur-1(ok1387) age (days of adulthood) OP50 D daf-2(e1368); nmur-1(ok1387); osm-6p::nmur-1 OP50 0 255 10 15 20 30 35 40 P - deaths onlyOP50 0 5 10 15 P - deaths onlyOP50 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint Sifoglu et al., Figure 5 A B daf-2(e1368) daf-2(e1368); nmur-1(ok1387)nmur-1(ok1387)wild type CS1800.0 1.0 0.2 0.4 0.6 0.8 fraction alive P - deaths onlyCS180 fraction alive 0.0 1.0 0.2 0.4 0.6 0.8 age (days of adulthood) 0 255 10 15 20 30 35 40 CS2429 P - deaths onlyCS2429 0 5 10 15 fraction alive fraction alive 0.0 1.0 0.2 0.4 0.6 0.8 0.0 1.0 0.2 0.4 0.6 0.8 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint nmur-1(ok1387) wild type 0.0 1.0 0.2 0.4 0.6 0.8 fraction aliveOP50 0 255 10 15 20 A daf-16(mu86) daf-16(mu86); nmur-1(ok1387) daf-16(mu86); daf-2(e1368); nmur-1(ok1387) wild type daf-16(mu86); nmur-1(ok1387)daf-2(e1368) daf-2(e1368); nmur-1(ok1387) age (days of adulthood) age (days of adulthood) daf-16(mu86); daf-2(e1368) B 0 255 10 15 20 30 35 40 0.0 1.0 0.2 0.4 0.6 0.8 fraction aliveOP50 0 5 10 15 OP50 P - deaths only 0 5 10 15 OP50 P - deaths only Sifoglu et al., Figure 6 C control 0 500 1000 1500 2000 2500 ins-6p::mCherry Fluorescence intensity (a.u.) nmur-1 (ok1387) D 0 500 1000 1500 2000 2500 daf-28p::mCherry Fluorescence intensity (a.u.) control nmur-1 (ok1387) ** .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint Sifoglu et al., Figure 7 daf-2 high activity nmur-1 daf-16 daf-2 low activity healthy pharynx and survival LPS structure daf-28 + other ILPs other ILPs? condition 1 condition 2 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 1 Table 1. Cumulative statistics of deaths on different bacteria Strain/Bacteria: All deaths Mean Lifespan ± SEM (Days) # Animals Observed/ Total Initial Animals (# Trials) P vs specified group (Logrank) P vs specified group (Wilcoxon) Fig Strain/Bacteria: P deaths # Animals Observed/ Total Initial Animals (# Trials) P vs specified group (Logrank) P vs specified group (Wilcoxon) Fig Effects of bacteria on wild-type P deaths Wild type (WT), OP50 12.5 ± 0.4 142/240 (3) - - 1A Wild type, OP50 74/240 (3) - - 1B Wild type (WT), CS180 14.2 ± 0.3 142/240 (3) 0.34WT/OP < 0.0001WT/OP 1A Wild type, CS180 22/240 (3) < 0.0001WT/OP < 0.0001WT/OP 1B WT, CS180 14.0 ± 0.3 186/320 (3) - - WT, CS180 26/320 (3) - - WT, CS2429 13.9 ± 0.3 187/320 (3) 0.94WT/CS180 0.64WT/CS180 WT, CS2429 80/320 (3) < 0.0001WT/CS180 < 0.0001WT/CS180 WT, CS180 13.7 ± 0.5 64/80 (1) - - 1H WT, CS180 17/80 (1) - - 1I WT, CS2198 12.2 ± 0.4 65/80 (1) 0.008WT/CS180 0.05WT/CS180 1H WT, CS2198 46/80 (1) < 0.0001WT/CS180 0.004WT/CS180 1I WT, CS2429 13.7 ± 0.6 61/80 (1) 0.56WT/CS180 0.77WT/CS180 1H WT, CS2429 35/80 (1) 0.004WT/CS180 0.02WT/CS180 1I nmur-1-dependent P deaths WT, OP50 11.9 ± 0.4 188/256 (1) - - 2A WT, OP50 73/240 (2) - - 2B nmur-1(ok1387), OP50 9.8 ± 0.4 137/176 (1) 0.0005WT/OP < 0.0001WT/OP 2A fln-2(ot611), OP50 34/230 (2) < 0.0001WT/OP < 0.0001WT/OP 2B fln-2(ot611), OP50 18.0 ± 0.6 49/70 (1) < 0.0001WT/OP < 0.0001WT/OP 2A WT, CS180 7/80 (1) - - 2B WT, CS180 13.1 ± 0.3 117/160 (1) - - 2A fln-2(ot611), CS180 0/80 (1) 0.01WT/CS 0.01WT/CS 2B nmur-1(ok1387), CS180 14.2 ± 0.4 64/80 (1) 0.03WT/CS 0.02WT/CS 2A WT, OP50 177/540 (4) - - 2D$ fln-2(ot611), CS180 12.8 ± 0.3 67/80 (1) 0.34WT/CS 0.50WT/CS 2A nmur-1(ok1387), OP50 244/540 (4) 0.0001WT/OP < 0.0001WT/OP 2D$ WT, CS180 22/320 (3) - - 2D$ nmur-1(ok1387), CS180 18/300 (3) 0.41WT/CS 0.38WT/CS 2D$ WT, OP50 12.4 ± 0.3 210/320 (2) - - 2F WT, OP50 108/400 (3) - - 2F nmur-1(lst1672), OP50 10.4 ± 0.3 232/320 (2) < 0.0001WT/OP < 0.0001WT/OP 2F nmur-1(lst1672), OP50 153/400 (3) < 0.0001WT/OP < 0.0001WT/OP 2F .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 2 Table 1. Continued Strain/Bacteria: All deaths Mean Lifespan ± SEM (Days) # Animals Observed/ Total Initial Animals (# Trials) P vs specified group (Logrank) P vs specified group (Wilcoxon) Fig Strain/Bacteria: P deaths # Animals Observed/ Total Initial Animals (# Trials) P vs specified group (Logrank) P vs specified group (Wilcoxon) Fig WT, OP50 10.9 ± 0.4 117/160 (2) - - 3A WT, OP50 177/540 (4) - - 3A$ nmur-1(ok1387), OP50 9.6 ± 0.4 120/160 (2) 0.05WT/OP 0.002WT/OP 3A nmur-1(ok1387), OP50 244/540 (4) 0.0001WT/OP < 0.0001WT/OP 3A$ daf-2(e1368), OP50 16.4 ± 0.8 94/160 (2) < 0.0001WT/OP < 0.0001WT/OP 3A daf-2(e1368), OP50 100/760 (4) < 0.0001WT/OP < 0.0001WT/OP 3A daf-2(e1368); nmur-1(ok1387), OP50 19.1 ± 0.7 113/160 (2) 0.03daf-2/OP 0.001daf-2/OP 3A daf-2(e1368); nmur-1(ok1387), OP50 56/540 (4) < 0.0001daf-2/OP < 0.0001daf-2/OP 3A WT, CS180 13.5 ± 0.3 106/160 (2) - - 3A WT, CS180 22/320 (3) - - 3A$ nmur-1(ok1387), CS180 14.4 ± 0.3 108/140 (2) 0.08WT/CS 0.04WT/CS 3A nmur-1(ok1387), CS180 18/300 (3) 0.41WT/CS 0.38WT/CS 3A$ daf-2(e1368), CS180 23.9 ± 0.5 104/160 (2) < 0.0001WT/CS < 0.0001WT/CS 3A daf-2(e1368), CS180 1/320 (3) < 0.0001WT/CS < 0.0001WT/CS 3A daf-2(e1368); nmur-1(ok1387), CS180 23.7 ± 0.4 105/160 (2) 0.54daf-2/CS 0.42daf-2/CS 3A daf-2(e1368); nmur-1(ok1387), CS180 4/320 (3) 0.29daf-2/CS 0.29daf-2/CS 3A WT, OP50 13.2 ± 0.3 145/240 (2) - - 3B WT, OP50 63/240 (2) - - 3B nmur-1(lst1672), OP50 10.7 ± 0.4 153/240 (2) < 0.0001WT/OP < 0.0001WT/OP 3B nmur-1(lst1672), OP50 89/240 (2) < 0.0001WT/OP < 0.0001WT/OP 3B daf-2(e1368), OP50 17.8 ± 0.7 89/240 (2) < 0.0001WT/OP < 0.0001WT/OP 3B daf-2(e1368), OP50 23/240 (2) 0.003WT/OP 0.004WT/OP 3B daf-2(e1368); nmur-1(lst1672), OP50 20.5 ± 0.5 93/320 (2) 0.004daf-2/OP < 0.0001daf-2/OP 3B daf-2(e1368); nmur-1(lst1672), OP50 20/320 (2) 0.009daf-2/OP 0.002daf-2/OP 3B WT, OP50 10.9 ± 0.4 158/220 (2) - - 3C WT, OP50 104/220 (2) - - 3C nmur-1(ok1387), OP50 10.2 ± 0.5 140/160 (1) 0.19WT/OP 0.004WT/OP 3C nmur-1(ok1387), OP50 88/160 (1) 0.20WT/OP 0.006WT/OP 3C daf-2(mu150), OP50 17.9 ± 0.8 130/420 (2) < 0.0001WT/OP < 0.0001WT/OP 3C daf-2(mu150), OP50 54/420 (2) < 0.0001WT/OP < 0.0001WT/OP 3C daf-2(mu150); nmur-1(ok1387), OP50 20.7 ± 0.7 161/420 (2) 0.004daf-2/OP 0.0001daf-2/OP 3C daf-2(mu150); nmur-1(ok1387), OP50 57/420 (2) 0.06daf-2/OP 0.006daf-2/OP 3C .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 3 Table 1. Continued Strain/Bacteria: All deaths Mean Lifespan ± SEM (Days) # Animals Observed/ Total Initial Animals (# Trials) P vs specified group (Logrank) P vs specified group (Wilcoxon) Fig Strain/Bacteria: P deaths # Animals Observed/ Total Initial Animals (# Trials) P vs specified group (Logrank) P vs specified group (Wilcoxon) Fig Rescue of nmur-1(ok1387) single mutants with nmur-1p::nmur-1 WT, OP50 11.5 ± 0.5 94/160 (1) - - 4A WT, OP50 120/336 (2) - - 4A nmur-1(ok1387), OP50 10.0 ± 0.4 136/160 (1) 0.002WT/OP < 0.0001WT/OP 4A nmur-1(ok1387), OP50 189/336 (2) 0.0002WT/OP < 0.0001WT/OP 4A nmur-1(ok1387); nmur-1p::nmur- 1, OP50 12.3 ± 0.4 105/160 (1) 0.22WT/OP < 0.0001ok1387 0.05WT/OP < 0.0001ok1387 4A nmur-1(ok1387); nmur-1p::nmur- 1, OP50 120/336 (2) 0.02WT/OP < 0.0001ok1387 0.04WT/OP < 0.0001ok1387 4A Rescue of nmur-1(ok1387) single mutants with osm-6p::nmur-1 WT, OP50 11.0 ± 0.4 91/176 (1) - - 4B WT, OP50 129/336 (2) - - 4B nmur-1(ok1387), OP50 9.5 ± 0.3 118/165 (1) 0.0008WT/OP < 0.0001WT/OP 4B nmur-1(ok1387), OP50 178/325 (2) < 0.0001WT/OP < 0.0001WT/OP 4B nmur-1(ok1387); osm-6p::nmur-1, OP50 10.5 ± 0.4 90/176 (1) 0.41WT/OP 0.01ok1387 0.35WT/OP 0.0002ok1387 4B nmur-1(ok1387); osm-6p::nmur-1, OP50 89/336 (2) 0.006WT/OP < 0.0001ok1387 0.005WT/OP < 0.0001ok1387 4B Rescue of daf-2(e1368); nmur-1(ok1387) double mutants with nmur-1p::nmur-1 WT, OP50 11.3 ± 0.5 92/160 (1) - - 4C WT, OP50 120/416 (3) - - 4C daf-2(e1368), OP50 17.2 ± 1.0 71/160 (1) < 0.0001WT/OP < 0.0001WT/OP 4C daf-2(e1368), OP50 55/416 (3) < 0.0001WT/OP < 0.0001WT/OP 4C daf-2(e1368); nmur-1(ok1387), OP50 19.8 ± 0.8 75/160 (1) 0.15daf-2/OP 0.005daf-2/OP 4C daf-2(e1368); nmur-1(ok1387), OP50 55/496 (3) 0.007daf-2/OP 0.0002daf-2/OP 4C daf-2(e1368); nmur-1(ok1387); nmur-1p::nmur- 1, OP50 17.0 ± 0.8 74/160 (1) 0.45daf-2/OP 0.01ok1387* 0.95daf-2/OP 0.002ok1387* 4C daf-2(e1368); nmur-1(ok1387); nmur-1p::nmur- 1, OP50 50/496 (3) 0.06daf-2/OP 0.6ok1387* 0.22daf-2/OP 0.05ok1387* 4C Rescue of daf-2(e1368); nmur-1(ok1387) double mutants with osm-6p::nmur-1 WT, OP50 11.2 ± 0.3 183/336 (2) - - 4D WT, OP50 98/336 (2) - - 4D daf-2(e1368), OP50 16.9 ± 0.6 150/336 (2) < 0.0001WT/OP < 0.0001WT/OP 4D daf-2(e1368), OP50 41/336 (2) 4D daf-2(e1368); nmur-1(ok1387), OP50 18.8 ± 0.5 164/336 (2) 0.04daf-2/OP 0.0003daf-2/OP 4D daf-2(e1368); nmur-1(ok1387), OP50 33/336 (2) 0.05daf-2/OP 0.01daf-2/OP 4D .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 4 Table 1. Continued Strain/Bacteria: All deaths Mean Lifespan ± SEM (Days) # Animals Observed/ Total Initial Animals (# Trials) P vs specified group (Logrank) P vs specified group (Wilcoxon) Fig Strain/Bacteria: P deaths # Animals Observed/ Total Initial Animals (# Trials) P vs specified group (Logrank) P vs specified group (Wilcoxon) Fig daf-2(e1368); nmur-1(ok1387); osm-6p::nmur-1, OP50 15.8 ± 0.7 130/336 (2) 0.33daf-2/OP 0.006ok1387* 0.05daf-2/OP < 0.0001ok1387* 4D daf-2(e1368); nmur-1(ok1387); osm-6p::nmur-1, OP50 21/336 (2) 0.16daf-2/OP 0.65ok1387* 0.08daf-2/OP 0.48ok1387* 4D LPS-dependence WT, CS180 13.9 ± 0.3 172/320 (3) - - 5A$$ WT, CS180 22/320 (3) - - 5A$$ nmur-1(ok1387), CS180 14.7 ± 0.3 185/300 (3) 0.06WT/CS 0.02WT/CS 5A$$ nmur-1(ok1387), CS180 18/300 (3) 0.41WT/CS 0.38WT/CS 5A$$ daf-2(e1368), CS180 23.9 ± 0.5 104/160 (2) < 0.0001WT/CS < 0.0001WT/CS 5A$$ daf-2(e1368), CS180 1/320 (3) < 0.0001WT/CS < 0.0001WT/CS 5A$$ daf-2(e1368); nmur-1(ok1387), CS180 23.7 ± 0.4 105/160 (2) 0.54daf-2/CS 0.54daf-2/CS 5A$$ daf-2(e1368); nmur-1(ok1387), CS180 4/320 (3) 0.29daf-2/CS 0.29daf-2/CS 5A$$ WT, CS2429 14.6 ± 0.3 126/240 (2) - - 5B WT, CS2429 87/400 (3) - - 5B nmur-1(ok1387), CS2429 12.9 ± 0.3 158/220 (2) 0.005WT/CS24 0.0004WT/CS24 5B nmur-1(ok1387), CS2429 127/380 (3) 0.0002WT/CS24 < 0.0001WT/CS24 5B daf-2(e1368), CS2429 25.4 ± 0.7 58/82 (1) < 0.0001WT/CS24 < 0.0001WT/CS24 5B daf-2(e1368), CS2429 33/400 (3) < 0.0001WT/CS24 < 0.0001WT/CS24 5B daf-2(e1368); nmur-1(ok1387), CS2429 26.7 ± 0.6 50/80 (1) 0.55daf-2/CS24 0.27daf-2/CS24 5B daf-2(e1368); nmur-1(ok1387), CS2429 16/400 (3) 0.005daf-2/CS24 0.004daf-2/CS24 5B daf-16-dependence WT, OP50 11.6 ± 0.5 136/176 (1) - - 6A,B WT, OP50 138/352 (2) - - 6A,B nmur-1(ok1387), OP50 9.8 ± 0.4 138/176 (1) 0.003WT/OP < 0.0001WT/OP 6A nmur-1(ok1387), OP50 149/352 (2) 0.02WT/OP 0.0002WT/OP 6A daf-16(mu86), OP50 9.1 ± 0.3 143/176 (1) < 0.0001WT/OP 0.0001WT/OP 6A daf-16(mu86), OP50 140/352 (2) 0.08WT/OP 0.0007WT/OP 6A daf-16(mu86); nmur-1(ok1387), OP50 9.2 ± 0.3 142/176 (1) 0.50daf-16/OP 0.83daf-16/OP 6A,B daf-16(mu86); nmur-1(ok1387), OP50 149/352 (2) 0.89daf-16/OP 0.75daf-16/OP 6A,B .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint 5 Table 1. Continued Strain/Bacteria: All deaths Mean Lifespan ± SEM (Days) # Animals Observed/ Total Initial Animals (# Trials) P vs specified group (Logrank) P vs specified group (Wilcoxon) Fig Strain/Bacteria: P deaths # Animals Observed/ Total Initial Animals (# Trials) P vs specified group (Logrank) P vs specified group (Wilcoxon) Fig daf-2(e1368), OP50 18.4 ± 0.7 109/176 (1) < 0.0001WT/OP < 0.0001WT/OP 6B daf-2(e1368), OP50 58/352 (2) < 0.0001WT/OP < 0.0001WT/OP 6B daf-2(e1368); nmur-1(ok1387), OP50 21.6 ± 0.6 116/176 (1) 0.008daf-2/OP 0.0004daf-2/OP 6B daf-2(e1368); nmur-1(ok1387), OP50 43/352 (2) 0.02daf-2/OP 0.003daf-2/OP 6B daf-16(mu86); daf-2(e1368), OP50 8.9 ± 0.3 120/176 (1) 0.70daf-16/OP 0.44daf-16/OP 6B daf-16(mu86); daf-2(e1368), OP50 146/352 (2) 0.22daf-16/OP 0.27daf-16/OP 6B daf-16(mu86); daf-2(e1368); nmur-1(ok1387), OP50 9.0 ± 0.3 140/176 (1) 0.78daf-16/OP 0.88daf-16/OP 6B daf-16(mu86); daf-2(e1368); nmur-1(ok1387), OP50 161/352 (2) 0.19daf-16/OP 0.35daf-16/OP 6B .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.15.670582doi: bioRxiv preprint