UPR^ER–immunity axis acts as physiological food evaluation system that promotes aversion behavior in sensing low-quality food

Reviewer #1 (Public Review):

Summary:
This manuscript by Liu et al explores the role of the UPR and immune regulators in the evaluation of nutritional quality in C. elegans. They identify neuronal UPR activation and the MAPK PMK-1 as key responders to low food quality. In particular, the data suggest that these pathways are activated by low levels of vitamin C synthesis that result from the low sugar levels present in heat-killed E. coli.

Strengths:
The results are intriguing and expand our understanding both of physiological food evaluation systems, and of the known roles of stress response pathways in organismal physiology. The authors use a range of techniques, encompassing imaging, metabolomic analysis, gene expression analysis, and behavioural assays, to support their claims.

Weaknesses:
There is limited mechanistic analysis in the study. In particular, how does low vitamin C trigger UPR activation? This is an intriguing finding that, if followed up, could potentially reveal a novel mechanism of UPR activation. In addition, how is the activation of the PMK-1 pathway driven by/coordinated with UPR activation? The data in some figures is not as convincing as it could be: the magnitude of the effect size is small in the supplementation experiments, and the statistical tests used are not always appropriate to enable multiple comparisons.

Reviewer #2 (Public Review):

Summary:
In this work, the authors aim to better understand how C. elegans detects and responds to heat-killed (HK) E. coli, a low-quality food. They find that HK food activates two canonical stress pathways, ER-UPR, and innate immunity, in the nervous system to promote food aversion. Through the creative use of E. coli genetics and metabolomics, the authors provide evidence that the altered carbohydrate content of HK food is the trigger for the activation of these stress responses and that supplementation of HK food with sugars (or their biosynthetic product, vitamin C), reduces stress pathway induction and food avoidance. This work makes a valuable addition to the literature on metabolite detection as a mechanism for the evaluation of nutritional value; it also provides some new insight into the physiologically relevant roles of well-known stress pathways in modulating behavior.

Strengths:
-The work addresses an important question by focusing on understanding how the nervous system evaluates food quality and couples this with behavioral change.
-The work takes full advantage of the tools available in this powerful system and builds on extensive previous studies on feeding behavior and stress responses in C. elegans.
-Creative use of E. coli genetics and metabolite profiling enabled the identification of carbohydrate metabolism as a candidate source of food-quality signals.
-For the most part, the studies are rigorous and logically designed, providing good support for the authors' model.

Weaknesses:
-It is not clear how the mechanism identified here is connected to previously described, related processes. In particular, it is not clear whether this mechanism has a role in the detection of other low-quality foods. Further, the specificity of the ability of sugar/vitamin C to suppress stress pathway induction is unclear (i.e., does sugar/vitamin C have any effect on the activation of these pathways through other means?). Additionally, the relationship of this pathway to the vitamin B2-sensing mechanism previously described by the senior author is unclear. These issues do not weaken confidence in the authors' conclusions, but they do reduce the potential significance of the work.

-The authors claim that the induction of the innate immune pathway reporter irg-5::GFP is "abolished" in pmk-1(RNAi) animals, but Figure S2K seems to show a clear GFP signal when these animals are fed HK-OP50. Similarly, the claim that feeding WT animals HK-OP50 enriches phospho-PMK-1 levels (Fig 2E) is unconvincing - only one western blot is shown, with no quantification, and there is a smear in the critical first lane.

-The rationales for some of the paper's hypotheses could be improved. For example, the rationale for screening the E. coli mutant library is that some mutants, when heat-killed, may be missing a metabolite that induces the ER-UPR. A more straightforward hypothesis might be that some mutant E. coli strains aberrantly induce the ER-UPR when *not* heat-killed, because they are missing a metabolite that prevents stress pathway induction. This is not in itself a major concern, but it would be useful for the authors to provide a rationale for their hypothesis.

-The authors do not provide any explanation for some unexpected results from the E. coli screen. Earlier in the paper, the authors found that innate immune signaling is downstream of ER-UPR activation. However, of the 20 E. coli mutants that, when heat-killed, "did not induce... the UPR-ER reporter," 9 of them still activate the innate immune response. This seems at odds with the authors' simple model since it suggests that low-quality food can induce innate immune signaling independently of the ER-UPR. Further, only one of the 9 has an effect on behavior, even though failure to activate the innate immune pathway might be expected to lead to a behavioral defect in all of these.

-In a number of places, the writing style can make the authors' arguments difficult to follow.

-Some of the effect sizes observed by the authors are exceedingly small (e.g, the suppression of hsp-4::gfp induction by sugar supplementation in Figs 3C-E), raising some concern about the biological significance of the effect.

-In some cases, there is a discrepancy between the fluorescence images and their quantitation (e.g., Figure 3E, where the effect of glucose on GFP fluorescence seems much stronger in the image than in the graph).

Reviewer #3 (Public Review):

Summary:
Animals can evaluate food quality in many ways. In contrast to the rapid sensory evaluation with smell and taste, the mechanism of slow nutrient sensation and its impact on food choice is unexplored. The authors utilize C. elegans larvae and their bacterial food as an elegant model to tackle this question and reveal the detailed molecular mechanism to avoid nutrient-poor foods.

Strengths:
The strength of this study is that they identified the molecular identities of the critical players in bacterial food and C. elegans using unbiased approaches, namely metabolome analysis, E. coli mutant screening, and RNA sequencing. Furthermore, they strengthen their findings by thorough experiments combining multiple methods such as genetics, fluorescent reporter analysis, and Western blot.

Weaknesses:
The major caveat of this study is the reporter genes. The transcriptional reporters were used to monitor the UPRER and immune responses in the intestine of C. elegans. However, their tissue-specific rescue experiments suggest that the genes in the UPRER and immune response function in the neurons. Thus, we should carefully interpret the results of the reporter genes.

Overall, this work provides convincing data to support their model. In the C. elegans field, the behaviors of larvae are not well studied compared to adults. This work will pose an interesting question about the difference between larvae and adults in nutrition sensing in C. elegans and provide a framework and candidate molecules to be studied in other organisms.