Acute avoidance of hydrogen sulfide is modulated by external and internal states in C. elegans

Reviewer #1 (Public Review):

Summary:
This paper sets out to achieve a deeper understanding of the effects of hydrogen sulfide on C. elegans behavior and physiology, with a focus on behavior, detection mechanism(s), physiological responses, and detoxification mechanisms.

Strengths:
The paper takes full advantage of the experimental tractability of C. elegans, with thorough, well-designed genetic analyses.
Some evidence suggests that H2S may be directly detected by the ASJ sensory neurons.
The paper provides interesting and convincing evidence for complex interactions between responses to different gaseous stimuli, particularly an antagonistic role between H2S and O2 detection/response.
Intriguing roles for mitochondria and iron homeostasis are identified, opening the door to future studies to better understand the roles of these components and processes.

Weaknesses:
The claim that worms' behavioral responses to H2S are mediated by direct detection is incompletely supported. While a role for the chemosensory neuron ASJ is implicated, it remains unclear whether this reflects direct detection. Other possibilities, including indirect effects of ASJ and the guanylyl cyclase daf-11 on O2 responses, are also consistent with the authors' data.

The role of H2S-mediated damage in behavioral responses, particularly when detoxification pathways are disrupted, remains unclear.

The findings of the paper are somewhat disjointed, such that a clear picture of the relationships between H2S detection, detoxification mechanisms, mitochondria, and iron does not emerge from these studies. Most importantly, the relative roles of H2S detection and integration, vs. general and acute mitochondrial crisis, in generating behavioral responses are not convincingly resolved.

Reviewer #2 (Public Review):

Summary:

H2S is a gas that is toxic to many animals and causes avoidance in animals such as C. elegans. The authors show that H2S increases the frequency of turning and the speed of locomotion. The response was shown to be modulated by a number of neurons and signaling pathways as well as by ambient oxygen concentrations. The long-term adaptation involved gene expression changes that may be related to iron homeostasis as well as the homeostasis of mitochondria.

Strengths:

Overall, the authors provide many pieces that will be important for solving how H2S signals through neuronal circuits to change gene expression and physiological programs. The experiments rely mostly on a behavioral assay that measures the increase of locomotion speed upon exposure to H2S. This assay is then combined with manipulations of environmental factors, different wild-type strains, and mutants. The mutants analyzed were obtained as candidates from the literature and from transcriptional profiling that the authors carried out in worms that were exposed to H2S. These studies imply several genetic signaling pathways, some neurons, and metabolism-related factors in the response to H2S. Hence the data provided should be useful for the field.

Weaknesses:

On the other hand, many important aspects of the underlying mechanisms remain unsolved and the reader is left with many loose ends. For example, it is not clear how H2S is actually sensed, how sensory neurons are activated and signal to downstream circuits, and what the role of ciliated and RMG neurons is in this circuit. It remains unclear how signals lead to gene expression and physiological changes such as metabolic rewiring. Solving all this would clearly be beyond the scope of a single manuscript. Yet, the manuscript also does not focus on understanding one of these central aspects and rather is all over the place, which makes it harder to understand for readouts that are not in this core field. Multiple additional methods and approaches exist to dig deeper into these mechanisms in the future, such as neuronal calcium imaging, optogenetics, and metabolic analysis. To generate a story that will be interesting to a broad readership substantial additional experimentation would be required. Further, in the current manuscript, it is often difficult to understand the rationales of the experiments, why they were carried out, and how to place them into a context. This could be improved in terms of documentation, narration/explanation, and visualization.

Reviewer #3 (Public Review):

Summary:
The manuscript explores the behavioral responses of C. elegans to hydrogen sulfide, which is known to exert remarkable effects on animal physiology in a range of contexts. The possibility of genetic and precise neuronal dissection of responses to H2S motivates the study of responses in C. elegans. The manuscript is well-written in communicating the complex physiology around C. elegans behavioral responses to H2S and in appropriately citing prior and related relevant work.

There are three parts to the manuscript.

In the first, an immediate behavioral response-increased locomotory rate-upon exposure to H2S is characterized. The experimental conditions are critical, and data are obtained from exposure of animals to 150ppm H2S at 7% O2. The authors provide evidence that this is a chemosensory response to H2S, showing a requirement for genes encoding components of the cilia apparatus and implicating a role for tax-4 and daf-11. Neuron-specific rescue in the ASJ neurons suggests the ASJ neurons contribute to the response to H2S. One caveat is that previous work has shown that the dauer-constitutive phenotype of daf-11 mutants can be suppressed by ASJ ablation, suggesting that there may be pervasive changes in animal nervous system signaling that are ASJ-dependent in daf-11 mutants, which may indirectly alter chemosensory responses to H2S. More direct methods to assess whether ASJ senses H2S, e.g. using calcium imaging, would better assess a direct role for the ASJ neurons in a behavioral response to H2S. The authors also point out interesting parallels between the response to H2S and CO2 though provide some genetic data separating the two responses. Importantly, the authors note that when aerotaxis (O2-sensing and movement) in the presence of bacterial food is intact, as in npr-1 215F animals, the response to H2S is abrogated. Mutation in gcy-35 in the npr-1 215F background restores the H2S chemosensory response.

There is a second part of the paper that conducts transcriptional profiling of the response to H2S that corroborates and extends prior work in this area.

The final part of the paper is the most intriguing, but for me, also the most problematic. The authors examine how H2S-evoked locomotory behavioral responses are affected in mutants defective in the stress and detoxification response to H2S, most notably hif-1. Prior genetic studies have established the pathways leading to HIF-1 activation/stabilization, as well as potential downstream mechanisms. The authors conduct logical genetic analysis to complement studies of the hif-1 mutant and in part motivated by their transcriptional profiling studies, examine the role of iron sequestration/free iron in the locomotory response to H2S, and further speculate on how the behavior of mutants defective in mitochondrial function might be affected by exposure to H2S.

In some regard, this part of the manuscript is interesting because the analysis begins to connect how the behavior of an animal to a toxic compound is affected by mutations that affect sensitivity to the toxic compound. However, what is unclear is what is being studied at this point. In the context, of noting that H2S at 150ppm is known to be lethal, its addition to mutants clearly sensitized to its effects would be anticipated to have pervasive effects on animal physiology and nervous system function. The authors note that the continued increased locomotion of wild-type animals upon H2S exposure might be due to the byproducts of detoxification or the detrimental effects of H2S. The latter explanation seems much more likely, in which case what one may be observing is the effects of general animal sickness, or even a bit more specifically, neuronal dysfunction in the presence of a toxic compound, on locomotion. As such, what is unclear is what conclusions can be taken away from this part of the work.

Strengths:
1. Characterization of a motor behavior response to H2S
2. Transcriptional profiling of the response to H2S corroborating prior work.

Weaknesses:
Unclear significance and experimental challenges regarding the study of locomotory responses to animals sensitized to the toxic effects of H2S under exposure to H2S.