Olfactory basis for essential amino acid perception during foraging in Caenorhabditis elegans

Reviewer #1 (Public review):

Summary:

Siddiqui et al., investigate the question of how bacterial metabolism contributes to the attraction of C. elegans to specific bacteria. They show that C. elegans prefers three bacterial species when cultured in a leucine-enriched environment. These bacterial species release more isoamyl alcohol, a known C. elegans attractant, when cultured with leucine supplement than without leucine supplement. The study shows correlative evidence that isoamyl alcohol is produced from leucine by the Ehrlich pathway. In addition, they show that SRD-12 is likely a receptor for isoamyl alcohol because a null mutant of this receptor exhibits lower chemotaxis to isoamyl alcohol and lower preference for leucine-enriched bacteria.

Strengths:

(1) This study takes a creative approach to examine the question of what specific volatile chemicals released by bacteria may signify to C. elegans by examining both bacterial metabolism and C. elegans preference behavior. Although C. elegans has long been known to be attracted to bacterial metabolites, this study may be one of the first to examine the role of a specific bacterial metabolic pathway in mediating attraction.

(2) A strength of the paper is the identification of SRD-12 as a likely receptor for isoamyl alcohol. The ligands for very few olfactory receptors have been identified in C. elegans and so this is a significant addition to the field. The srd-12 null mutant strain will likely be a useful reagent for many labs examining olfactory and foraging behaviors.

Weaknesses:

(1) The authors write that the leucine metabolism via the Ehrlich pathway is required for the production of isoamyl alcohol by three bacteria (CEent1, JUb66, BIGb0170), but their evidence for this is correlation and not causation. They write that the gene ilvE is a bacterial homolog of the first gene in the yeast Ehrlich pathway (it would be good to include a citation for this) and that the gene is present in these three bacterial strains. In addition, they show that this gene, ilvE, is upregulated in CEent1 bacteria upon exposure to leucine. To show causation, they need to knockout ilvE from one of these strains, show that the bacteria does not have increased isoamyl alcohol production when cultured on leucine, and that the bacteria is no longer attractive to C. elegans.

(2) The authors examine three bacterial strains that C. elegans showed increased preference when grown with leucine supplementation vs. without leucine supplementation. However, there also appears to be a strong preference for another strain, JUb0393, when grown on plus leucine (Figure 1B). It would be good to include statistics and criteria for selecting the three strains.

3. Although the behavioral evidence that srd-12 gene encodes a receptor for isoamyl alcohol is compelling, it does not meet the standard for showing that it is an olfactory receptor in C. elegans. To show it is indeed a likely receptor one or more of the following should be done:
(a) Calcium imaging of AWC neurons in response to isoamyl alcohol in the receptor mutant with the expectation that the response would be reduced or abolished in the mutant compared to wildtype.
(b)"A receptor swap" experiment where the SRD-12 receptor is expressed in AWB repulsive neuron in SRD-12 receptor mutant background with the expectation that with receptor swap C. elegans will now be repulsed from isoamyl alcohol in chemotaxis assays (experiment from Sengupta et al., 1996 odr-10 paper).

(4) The authors conclude that C. elegans cannot detect leucine in chemotaxis assays. It is important to add the method for how leucine chemotaxis assay was done in order to interpret these results. Because leucine is not volatile if leucine is put on the plates immediately before the worms are added (as in a traditional odor chemotaxis assay), there is no leucine gradient for the worm to detect. It would be good to put leucine on the plate several hours before worms are introduced so worms have the possibility to be able to detect the gradient of leucine (for example, see Wakabayashi et al., 2009).

(5) The bacterial preference assay entitled "odor-only assay" is a misleading name. In the assay, C. elegans is exposed to both volatile chemicals (odors) and non-volatile chemicals because the bacteria are grown on the assay plate for 12 hours before the worms are introduced to the assay plate. In that time, the bacteria is likely releasing non-volatile metabolites into the plate which may affect the worm's preference. A true odor-only assay would have the bacteria on the lid and the worms on the plate.

(6) The findings of the study should be discussed more in the context of prior literature. For example, AWC neurons have been previously shown to be involved in bacterial preference (Harris et al., 2014; Worthy et al., 2018). In addition, CeMbio bacterial strains (the strains examined in this study) have been previously shown to release isoamyl alcohol (Chai et al. 2024).

Reviewer #2 (Public review):

Summary:

Siddiqui et al. show that C. elegans prefers certain bacterial strains that have been supplemented with the essential amino acid (EEA) leucine. They convincingly show that some leucine enriched bacteria stimulate the production of isoamyl alcohol (IAA). IAA is an attractive odorant that is sensed by the AWC. The authors an identify a receptor, SRD-12, that is expressed in the AWC chemosensory neurons and is required for chemotaxis to IAA. The authors propose that IAA is a predominant olfactory cue that determines diet preference in C. elegans. Since leucine is an EAA, the authors propose that worm IAA sensing allows the animal provides a proxy mechanism to identify EAA rich diets.

Strengths:

The authors propose IAA as a predominant olfactory cue that determines diet preference in C. elegans providing molecular mechanism underlying diet selection. They show that wild isolates of C. elegans have a strong chemotactic response to IAA indicating that IAA is an ecologically relevant odor for the worm. The paper is well written, and the presented data are convincing and well organized. This is an interesting paper that connects chemotactic response with bacterially produced odors and thus provides an understanding of how animals adapt their foraging behavior through the perception of molecules that may indicate the nutritional value.

Weaknesses:

Major:

While I do like the way the authors frame C. elegans IAA sensing as mechanisms to identify leucine (EAA) rich diets it is not fully clear whether bacterial IAA production is a proxy for bacterial leucine levels.

(1) Can the authors measure leucine (or other EAA) content of the different CeMbio strains? This would substantiate the premise in the way they frame this in the introduction. While the authors convincingly show that leucine supplementation induces IAA production in some strains, it is not clear if there are lower leucine levels in the different in non-preferred strains.

(2) It is not clear whether the non-preferred bacteria in Figure 1A and 1B have the ability to produce IAA. To substantiate the claim that C. elegans prefers CEent1, JUb66, and BIGb0170 due to their ability to generate IAA from leucine, it would measure IAA levels in non-preferred bacteria (+ and - leucine supplementation). If the authors have these data it would be good to include this.

(3) The authors would strengthen their claim if they could show that deletion or silencing ilvE enzyme reduces IAA levels and eliminates the increased preference upon leucine supplementation.

(4) While the three preferred bacteria possess the ilvE gene, it is not clear whether this enzyme is present in the other non-preferred bacterial strains. As far as I know, the CeMbio strains have been sequenced so it should be easy to determine if the non-preferred bacteria possess the capacity to make IAA. Does the expression of ilvE in e.g. E. coli increase its preference index or are the other genes in the biosynthesis pathway missing?

(5) It is strongly implied that leucine-rich diets are beneficial to the worm. Do the authors have data to show the effect on leucine supplementation on C. elegans healthspan, life-span or broodsize?

Other comments:

Page 6. Figure 2c. While the authors' conclusions are correct based on AWC expts. it would be good at this stage to include the possibility that odors that enriched in the absence of leucine may be aversive.

Page 6. IAA increases 1.2-4 folds upon leucine supplementation. If the authors perform a chemotaxis assay with just IAA with 1-2-4 fold differences do you get the shift in preference index as seen with the bacteria? i.e. is the difference in IAA concentration sufficient to explain the shift in bacterial PI upon leucine supplementation? Other attractants such as Acetoin and isobutanol go up in -Leu conditions.

Page 14-15. The authors identify a putative IAA receptor based on expression studies. I compliment the authors for isolating two CRISPR deletion alleles. They show that the srd-12 mutants have obvious defects in IAA chemotaxis. Very few ligand-odorant receptors combinations have been identified so this is an important discovery. CenGen data indicate that srd-12 is expressed in a limited set of neurons. Did the authors generate a reporter to show the expression of srd-12? This is a simple experiment that would add to the characterization of the SRD-12 receptor. Rescue experiments would be nice even though the authors have independent alleles. To truly claim that SRD-12 is the ligand for IAA and activates the AWC neurons would require GCamp experiments in the AWC neuron or heterologous expression system. I understand that GCamp imaging might not be part of the regular arsenal of the lab but it would be a great addition (even in collaboration with one of the many labs that do this regularly). Comparing AWC activity using GCaMP in response IAA-producing bacteria with high leucine levels in both wild-type and SRD-12-deficient backgrounds, would further support their narrative. I leave that to the authors.

Minor:

Page 4 "These results suggested that worms can forage for diets enriched in specific EAA, leucine...." More precise at this stage would be to state " These results indicated that worms can forage for diets supplemented with specific EAA...".

Page 5."these findings suggested that worms not only rely on odors to choose between two bacteria but also to find leucine enriched bacteria" This statement is not clear to me and doesn't follow the data in Fig. S2. Preferred diets in odorant assays are the IAA producing strains.

Page 5. Figure S2A provides nice and useful data that can be part of the main Figure 1.

Reviewer #3 (Public review):

Summary:

The authors first tested whether EAA supplementation increases olfactory preference for bacterial food for a variety of bacterial strains. Of the EAAs, they found only leucine supplementation increased olfactory preference (within a bacterial strain), and only for 3 of the bacterial strains tested. Leucine itself was not found to be intrinsically attractive.

They determined that leucine supplementation increases isoamyl alcohol (IAA) production in the 3 preferred bacterial strains. They identify the biochemical pathway that catabolizes leucine to IAA, showing that a required enzyme for this pathway is upregulated upon supplementation.

Consistent with earlier studies, they find that AWC olfactory neuron is primarily responsible for increased preference for IAA-producing bacteria.

Testing volatile compounds produced by bacteria and identified by GC/MS, and identified several as attractive, most of them require AWC for the full effect. Adaptation assays were used to show that odorant levels produced by bacterial lawns were sufficient to induce olfactory adaptation, and adaptation to IAA reduced chemotaxis to leucine-supplemented lawns. They then showed that IAA attractiveness is conserved across wild strains, while other compounds are more variable, suggesting IAA is a principal foraging cue.

Finally, using the CeNGEN database, they developed a list of candidate IAA receptors. Using behavioral tests, they show that mutation of srd-12 greatly impairs IAA chemotaxis without affecting locomotion or attraction to another AWC-sensed odor, PEA.

Comments

This study will be of great interest in the field of C. elegans behavior, chemical senses and chemical ecology, and understanding of the sensory biology of foraging.

Strengths:

The identification of a receptor for IAA is an excellent finding. The combination of microbial metabolic chemistry and the use of natural bacteria and nematode strains makes an extremely compelling case for the ecological and adaptive relevance of the findings.

Weaknesses:

AWC receives synaptic input from other chemosensory neurons, and thus could potentially mediate navigation behaviors to compounds detected in whole or in part by those neurons. Language concluding detection by AWC should be moderated (e.g. p9 "worms sense an extensive repertoire...predominantly using AWC") unless it has been demonstrated.

srd-12 is not exclusively expressed in AWC. Normally, cell-specific rescue or knockdown would be used to demonstrate function in a specific cell. The authors should provide such a demonstration or explain why they are confident srd-12 acts in AWC.

A comparison of AWC's physiological responses between WT and srd-12, preferably in an unc-13 background, would be nice. Even further, the expression of srd-12 in a different neuron type and showing that it confers responsiveness to IAA (in this case, inhibition) would be very convincing.