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. 2022 Sep 29;17(9):e0274226. doi: 10.1371/journal.pone.0274226

Distinct mechanisms underlie H2O2 sensing in C. elegans head and tail

Sophie Quintin 1,2,3,4,*, Théo Aspert 1,2,3,4, Tao Ye 1,2,3,4, Gilles Charvin 1,2,3,4
Editor: Elise A Kikis5
PMCID: PMC9521893  PMID: 36173997

Abstract

Environmental oxidative stress threatens cellular integrity and should therefore be avoided by living organisms. Yet, relatively little is known about environmental oxidative stress perception. Here, using microfluidics, we showed that like I2 pharyngeal neurons, the tail phasmid PHA neurons function as oxidative stress sensing neurons in C. elegans, but display different responses to H2O2 and light. We uncovered that different but related receptors, GUR-3 and LITE-1, mediate H2O2 signaling in I2 and PHA neurons. Still, the peroxiredoxin PRDX-2 is essential for both, and might promote H2O2-mediated receptor activation. Our work demonstrates that C. elegans can sense a broad range of oxidative stressors using partially distinct H2O2 signaling pathways in head and tail sensillae, and paves the way for further understanding of how the integration of these inputs translates into the appropriate behavior.

Introduction

Reactive oxygen species (ROS) are well-known to exert a dual effect, promoting aging and pathological conditions on the one hand and increasing organism resistance and longevity on the other hand [1]. Neurons are easily exposed to ROS, and many neurodegenerative diseases have been associated with oxidative stress [2]. However, exposure of C. elegans nematodes to a mild oxidative stress has been reported to be beneficial for neuron sensory function: micromolar doses of the ROS-inducing agent paraquat or of hydrogen peroxide (H2O2) improve the sensitivity in ASH polymodal neurons [3, 4], whereas millimolar doses of H2O2 reduce neuron response [3], likely inducing oxidative stress. Therefore, it is essential for nematodes to detect a broad range of H2O2 concentrations to preserve their cellular integrity.

Although the cellular response to oxidative stress has been extensively characterized (reviewed in [5]), little is known on how oxidants are actually perceived and which underlying molecular pathways are involved. A recent study has revealed the behavioral mechanisms which allow C. elegans to find a niche providing both food and protection from H2O2 [6]. In addition, light and H2O2 sensing were shown to be tightly connected in yeast [7] and in nematodes [8]. While it has been demonstrated that light is converted into an H2O2 signal in yeast [7], this question remains unanswered in nematodes. Notably, C. elegans can detect both H2O2 and light via the I2 pharyngeal neurons and responds to these stimuli by inhibition of feeding or by an avoidance behavior [8]. Initially described as interneurons, the I2 neurons proved to be primary sensory neurons [9] that are highly specialized in oxidative stress sensing [8], but were also shown to respond moderately to salt or odor [10]. However, although detection of a large spectrum of H2O2 is critical for nematodes, the range of H2O2 concentrations detected by I2 neurons has not been investigated, and the molecular mechanisms involved in H2O2 signaling are not well defined.

In addition, the nematode also possesses tail sensory neurons specialized in chemo-repulsion, called PHA/PHB (or phasmids, reviewed in [11]). In contrast to I2 neurons, these tail neurons can respond to many noxious stimuli [12] and trigger avoidance [13], but whether PHA neurons can sense H2O2 or light remains an open question.

H2O2 sensing in I2 neurons requires the function of the peroxiredoxin PRDX-2 [8], a highly conserved antioxidant enzyme whose role remains unclear. Peroxiredoxins (Prxs) belong to a family of thiol peroxidases which can reduce H2O2 in cells following the oxidation of one or two cysteines in their catalytic domain, and they are highly abundant from yeast [14, 15] to human cells, in which they represent approximately 1% of the total dry cellular mass [16, 17]. Oxidised Prxs are recycled in the reduced, active form, by thioredoxin [18]. At high H2O2 concentrations, Prxs become hyperoxidized, a form that has been shown to function as a molecular chaperone [19, 20]. Similarly, thioredoxins have been shown to have redox-independent function [21, 22]. As pivotal antioxidants, Prxs dysfunction has been associated with several pathologies [23], including cancer [24]. In budding yeast, the peroxiredoxin Tsa1 has a major role in maintaining the redox balance, and is massively induced upon oxidative stress as a direct target of the H2O2-sensing transcription factor Yap1 [25, 26]. In C. elegans, among three genes encoding peroxiredoxins, PRDX-2 is the only one whose depletion induces a phenotype and is considered as the major peroxiredoxin [27, 28]. PRDX-2 is expressed in many cell types including neurons, gut [2729], muscle and epithelial cells [8]. Although a global induction of prdx-2 mRNA expression has been reported upon treatment with the strong tBOOH oxidant [28], cells in which PRDX-2 expression is induced remain to be identified. Likewise, the question of a tissue-specific regulation of PRDX-2 by SKN-1, the closest ortholog of Yap1, has not been addressed.

Importantly, beyond their peroxidase activity, Prxs have long been proposed to act as intracellular H2O2 sensors, which influence cellular signaling [3032]. For example, the p38/MAPK signaling pathway, which controls adaptive mechanisms and/or cell fate decisions, is activated by H2O2 through Prxs in an evolutionary conserved manner [33, 34]. Like in mammalian or in drosophila cells, several studies in C. elegans indicate that PRDX-2 would relay H2O2 signaling, activating the downstream p38/PMK-1 pathway [3, 35, 36]. Notably, low doses of H2O2 potentiate the sensory response of ASH sensory neurons to glycerol through activation of the PRDX-2/p38/PMK-1 cascade [3]. Yet, whether this cascade is at play in I2 neurons to influence H2O2 perception has not been analyzed.

Here, we undertook a subcellular analysis of the peroxiredoxin PRDX-2 in C. elegans, focusing on its requirement in neuronal H2O2 sensing. Using a CRISPR knock-in line, we showed that PRDX-2 is present in many cells, among which several pairs of neurons: I2s in the head, PHAs in the tail and CANs in the body. Interestingly, upon an H2O2 challenge, an upregulation of PRDX-2 is observed only in the anterior gut and in the excretory pore, but not in neurons, suggesting that PRDX-2 might fulfill different functions, depending on the cell type where it is expressed. Using a microfluidic-based approach and real-time calcium imaging, we show that PHA neurons also respond to H2O2, with an even higher sensitivity than I2 neurons. Although H2O2 perception depends on prdx-2 function in both pairs of neurons, we uncovered that it relies on different gustatory receptors and downstream transducers: while dispensable in I2 neurons, the p38/MAPK kinase pathway contributes to the hypersensitivity of PHA neurons to H2O2. Interestingly, we uncovered that H2O2 sensing requires the same receptors as light sensing, and that PHA neurons respond to light—establishing a parallel between PHA and photosensory ASH neurons. Based on our work and on previous studies, we propose a molecular model of how H2O2 could trigger neuronal activation in I2 and PHA through a peroxiredoxin-mediated redox relay. Taken together, our data suggest that C. elegans can sense a broad range of oxidative stress using partially distinct H2O2 signaling pathways acting in head and tail sensillae.

Results

Expression pattern of PRDX-2::GFP and its variation upon H2O2 treatment

In budding yeast, the peroxiredoxin Tsa1 is massively induced upon H2O2 treatment [25, 26]. To gain insight into the tissue-specific expression of PRDX-2 upon oxidative stress, we first used the PRDX-2 reporter line generated by [29]. However, the level of expression of this strain varies a lot, and transgenics show many fluorescent aggregates ([29], S1 Fig), likely associated with transgene overexpression. Consistently, the strain displays a much stronger resistance to oxidative stress than wild-type animals (S1 Fig), suggesting that overexpressed PRDX-2 construct induces a higher H2O2 scavenging capacity in transgenics. Furthermore, it was impossible to identify PRDX-2-expressing cells in this strain, preventing the use of this strain in our study. For these reasons, we created a GFP knock-in line of PRDX-2, using the CRISPR-Cas9 technique [37]. A C-terminus GFP fusion targeting all PRDX-2 isoforms, comprising a linker, was engineered and inserted at the prdx-2 locus (Fig 1A). Three independent knock-in lines were obtained, sharing an identical expression pattern (Figs 1B and S1).

Fig 1. PRDX-2::GFP knock-in line expression pattern and its variation upon H2O2 treatment.

Fig 1

(A) Sketch depicting the PRDX-2::GFP knock-in strategy using CRISPR Cas9-mediated genome editing. The sgRNA target sequence (shown in red) was chosen upstream PRDX-2 STOP codon, to tag all prdx-2 isoforms. Black boxes indicate exons, white boxes untranslated regions. Bar, 100 bases pairs. The resulting last exon of the PRDX-2::GFP fusion protein is shown (right); note the insertion of a flexible linker between PRDX-2 and the GFP, to promote correct folding. After injection, three independent knock-in lines were recovered, sharing the same expression pattern. (B) Spinning-disc confocal projections of a representative PRDX-2::GFP knock-in animal, in 4 body regions (boxed in the worm sketch on top). PRDX-2::GFP expression is observed in the tip of the nose (e1, e3 epithelial cells), in pharyngeal muscle cells (pm), in body wall muscles (m), in the excretory pore cell (EPC), in proximal and distal gut cells (int1 and int9), and in several neuron pairs (I2, CAN, PHA/PHB). Note the different level of PRDX-2::GFP in I2 left and right neurons in panel B1. (C-G) An acute oxidative stress triggers an upregulation of PRDX-2 in the anterior gut, but not in neurons. (C-D) Spinning-disc confocal projections of control or H2O2-treated animals in the anterior gut (C) or in the head (D). (E-G) Quantification of fluorescence intensity in controls and in H2O2-treated animals in the int1 cell (E), in the EPC (F), and I2 neurons (G). Bars indicate mean and SD. ns, not significant, p>0.05; *p<0.05; ***p<0.001 (t test and Mann Whitney test). Scale bar, 20μm.

In the PRDX-2::GFP knock-in line, we detected a broad expression of PRDX-2 in various cell types, including the proximal and distal gut, muscles (body wall, vulval and pharyngeal), epithelial cells (vulva, nose tip, hypodermis) and I2 neurons (Fig 1B), as previously reported [8, 27, 28]. In addition, we observed for the first time PRDX-2 expression in the excretory pore cell (EPC), and in two other pairs of neurons; the tail phasmids (PHA/PHB, whose identity was ascertained by DiI staing, S1 Fig) and the excretory canal-associated neurons (CANs), located close to the vulva (Fig 1B). Consistent with this, a high number of prdx-2 transcripts was detected in CANs and in the EPC [38]. Therefore, we hypothesized that the knock-in line faithfully reflects the endogenous PRDX-2 expression, and characterized it further.

PRDX-2::GFP is induced in the anterior gut upon H2O2 treatment, but not in I2 neurons

We noticed that many PRDX-2-expressing cells are directly in contact with the environment, such as the EPC, the tip of the nose, the vulva, and neurons, which all possess terminations exposed outside. This pattern is strikingly reminiscent of that detected in animals carrying the HyPer H2O2 biosensor after H2O2 exposure [39]. Thus, PRDX-2 is expressed in cells in which environmental H2O2 penetrates more easily, suggesting a protective role of PRDX-2 in these cells as a peroxidase. Therefore, in the following, we wondered whether all PRDX-2-expressing cells respond similarly to an H2O2-induced oxidative stress.

To determine whether PRDX-2 expression changes upon oxidative stress, we exposed animals to different doses of H2O2, and PRDX-2::GFP expression was quantified in different cells after spinning-disc confocal acquisitions. We selected H2O2 concentrations inducing different physiological responses [8]. A higher PRDX-2 expression was detected in the anterior gut two hours after a 10mM H2O2 treatment, but not after a 1mM H2O2 treatment (Fig 1C and 1E). The fluorescence was specific to PRDX-2::GFP as H2O2-treated controls did not show a higher gut autofluorescence (S2 Fig). Similarly, PRDX-2 expression was only induced in the EPC at 10mM H2O2 (Figs 1C and 1F and S2). Thus, our data indicate a dose-dependent PRDX-2 induction—as it occurs at 10mM but not at 1mM. The origin of this difference could arise from the animal behavioral response: at 1mM H2O2 the pharyngeal pumping is strongly inhibited to prevent ingestion [8], thereby exposure of gut to H2O2. This could explain both the absence of PRDX-2 induction in the gut we report, and the unchanged level of prdx-2 mRNA reported [28], after a 1mM H2O2 treatment. In contrast, at 10mM H2O2, the nematode should retract back (avoidance response, [8]); but here, as trapped animals cannot escape, they likely swallow a certain amount of H2O2, exposing the foregut to a severe oxidative stress which could trigger PRDX-2 induction. A 10mM H2O2 treatment of 30min has been shown to induce hyperoxidation of over 50% total PRDX-2 in wild-type lysed worms [40] and this form persists after a 4h recovery period [27]. Although the induction we observed is only based on expression level and not on protein activity, we suggest that PRDX-2 could still scavenge H2O2 in the EPC and in the foregut, consistent with the reported protective role of intestinal PRDX-2 [27].

Among PRDX-2-expressing neurons, the I2 pair shows the highest expression and possesses terminations exposed to the outside [9]. Therefore, we focused on I2 neurons to investigate whether the neuronal level of PRDX-2 is affected upon H2O2 treatment. However, at both concentrations tested (1 and 10mM), the level of PRDX-2 in I2 neurons constantly remained unchanged after H2O2 treatment (Figs 1D and 1G and S2).

Thus, we observed an upregulation of PRDX-2::GFP in the anterior gut and in the EPC following an acute oxidative stress, but not in neurons. As Prxs belong to a homeostatic system, an upregulation was shown to be associated with a detoxification function in yeast [26]. By analogy, PRDX-2 might fulfill a peroxidase function in the foregut and in the EPC to protect the animal against environmental aggressions. In contrast, in I2 neurons, PRDX-2 would instead act in H2O2 sensing and/or signaling, as proposed [8, 28]. We conclude that the responses of PRDX-2 to oxidative stress are cellular context-dependent.

SKN-1 controls expression of PRDX-2 in the gut, but not in neurons

This prompted us to test whether PRDX-2 expression could rely on different subcellular regulations. By analogy with yeast, we wondered whether PRDX-2 would be controlled by the Yap1 nematode orthologue SKN-1 [5] in cells where PRDX-2 is induced upon oxidative stress, and asked whether such regulation occurs in other cells. We sought to test this hypothesis by inactivating the function of SKN-1 in the PRDX-2::GFP knock-in line. We first used the skn-1(zj15) allele, which specifically inactivates gut-specific isoforms, leaving neuronal isoforms unaffected [41]. In this mutant, the basal expression level of PRDX-2 is reduced in the foregut, but an induction persists after a 10mM H2O2 treatment (Fig 2C), suggesting that the neuronal isoforms could mediate this effect. Consistent with this, the RNAi-mediated knock-down of all skn-1 isoforms also triggered a reduction of PRDX-2::GFP signal in the anterior gut, and an absence of induction upon H2O2 treatment (Fig 2A, 2B and 2D). This result indicates that SKN-1 activity is essential to regulate PRDX-2 expression in the anterior gut, both at the basal level and under oxidative stress. In agreement with this, SKN-1 was found to bind to the prdx-2 promoter in chromatin immunoprecipitation experiments coupled with high-throughput DNA sequencing (ChIP-seq) [42] (S3 Fig).

Fig 2. SKN-1 function is required for PRDX-2 expression in the gut.

Fig 2

(A-B) Spinning-disc confocal projections of head and anterior gut of PRDX-2::GFP knock-in animals, in control RNAi (A) and in skn-1(RNAi) (B) animals. The right panel shows the foregut of a 10mM H2O2-treated animal in both genotypes. Note the low level of int1 PRDX-2::GFP fluorescence in skn-1(RNAi) animal. (C-D) Quantification of the int1 cell fluorescence intensity in control and after a 10mM H2O2-treatment, in skn-1(zj15) mutants (C) and in skn-1(RNAi) animals (D). Bars indicate mean and SD. ns, not significant, p>0.05; **p<0.01; ***p<0.001; ***p<0.0001 (t test, ANOVA). Scale bar, 20μm.

In contrast, in I2 neurons, no change in the PRDX-2 level was detected at the basal level or after H2O2 treatment compared to controls, neither in skn-1(zj15) mutants nor in skn-1(RNAi) animals (S2 Fig). With the limitation that RNAi efficiency may be lower in neurons [43], we suggest that additional transcription factor(s) might regulate prdx-2 expression in these neurons. Consistently, several transcription factors were reported to bind to the prdx-2 promoter by ChIP-seq [42] (S3 Fig).

We conclude that skn-1 accomplishes a cell autonomous regulation of prdx-2 in the intestine, but that this regulation may not occur in neurons. Taken together, our data suggest that PRDX-2 might exert a different function depending on the cell type: H2O2 detoxification in the foregut, triggered by SKN-1, vs. H2O2 perception or signaling in I2 neurons.

PHA neurons respond to H2O2 in a prdx-2-dependent manner

As PRDX-2 is expressed in other neurons than I2s, we asked whether PRDX-2 endows these neurons with oxidative stress sensing properties. We focused on PHA tail neurons, as they belong to the phasmid sensory sensilla and respond to many noxious stimuli [12]. We monitored PHA neuron activation by imaging calcium using the GCaMP3 fluorescent sensor [44] expressed under the flp-15 promoter, specific to I2 and PHA neurons [45].

To image calcium fluxes in neurons, L4 animals were trapped in microfluidic chambers and exposed to H2O2 (Figs 3A and S4), and their response was followed in 4D spinning-disc ultrafast acquisitions. This experimental setup, combined with semi-automated image analyses to quantify the mean fluorescence in neurons over time (Supplementary Information), confirmed the activation of I2 neurons upon a 1mM H2O2 treatment (Fig 3B and 3D and S1 Movie and S5 Fig), similar to that observed upon exposure to H2O2 vapor (ie. 8.82M, [8]). As PRDX-2 reproducibly shows an asymmetric expression level in I2L and I2R (Fig 1B and 1D and detailed description in [46]), each neuron was scored individually to take into account a putative left-right effect. However, this did not impact neuron response as no significant difference in the normalized response of left and right neurons was noticed (Fig 3B and 3L). Importantly, we observed that PHA neurons respond to 1mM H2O2 comparably to I2 neurons (Fig 3C, 3D and 3H and S2 Movie and S5 Fig). We then investigated whether PRDX-2 function was necessary for PHA response, using a strong prdx-2 loss of function mutant. In agreement with previous work [8], I2 neurons in prdx-2(gk169) mutants failed to respond to 1mM H2O2 (Fig 3E, 3F and 3H and S3 Movie and S5 and S6 Figs). Interestingly, we uncovered that PHA neuron response was severely impaired in prdx-2 mutants, although not completely abolished as in I2s (Fig 3E, 3G and 3H and S4 Movie and S5 and S6 Figs). From these observations, we conclude that both I2s and PHAs responses to oxidative stress require the function of the peroxiredoxin PRDX-2, but the residual response of PHA neurons suggests a lesser requirement of the antioxidant in tail neurons.

Fig 3. I2 and PHA neurons both respond to 1mM H2O2 in a prdx-2-dependent manner, but only PHA neurons respond to 10μM H2O2.

Fig 3

(A) Low-magnification DIC picture of L4 animals trapped in the microfluidic device used in all neuron recordings experiments. (B,C,F,G,J,K) Calcium response (visualized by GCaMP3) in I2R/I2L (B,F,J) and in PHAL/PHAR neurons (C,G,K) following H2O2 treatment (indicated by the red bar). Average curves show normalized neuron responses (time in seconds), in both left and right neurons (top and bottom curves). N, number of movies quantified for each genotype, in wild-type (B,C,J,K) and in prdx-2 mutants (F,G). (D,E,I) Bar graph showing the fraction of animals responding to the H2O2 stimulation in all experiments, classified as high (green), moderate (yellow) or absent (red) responses (see Methods). N, number of movies analyzed. (H,L) Quantification of the calcium response to H2O2 in I2 and PHA neurons in controls and in prdx-2 mutants at 1mM (H), and in I2L/R and PHAL/R controls, at 1mM and 10μM H2O2 (L). Bars represent the mean (N, number of movies) and error bars SD. ns, not significant, p>0.05; *p<0.05; ***p<0.001; ****p<0.0001 (Mann Whitney and Kruskal-Wallis tests). See corresponding S1S6 Movies and S5 Fig.

I2s and PHAs show differences in H2O2 sensitivity and in receptors involved

Given the putative role of PRDX-2 as an H2O2 sensor, the fact that there was a slight difference in PRDX-2 activity requirement in I2s and PHAs prompted us to analyze whether the head and tail neurons share the same sensitivity to H2O2. We thus tested whether I2 and PHA neurons exhibit a response to the very mild dose of 10μM H2O2, a dose which induces a less penetrant pharyngeal pumping inhibition (≈ 35% of animals) than that observed at 1mM (≈ 90% of animals, [8]). In our experiments, whereas I2 neurons failed to be activated in most animals at 10μM H2O2 (25/29, Fig 3I, 3J and 3L and S5 Movie and S5 Fig), PHA neurons responded in the vast majority of animals (25/27, Fig 3I), in a similar manner than at 1mM H2O2 (Fig 3K and 3L and S6 Movie and S5 Fig). In addition, PHA neurons response to 10μM H2O2 depends on PRDX-2, as prdx-2 mutants PHA neurons all fail to respond to this low dose (13/13, S7 Movie and S5 and S6 Figs). The requirement of PRDX-2 for PHA response to micromolar H2O2 suggests that PRDX-2 is unlikely to transmit the H2O2 signal under its hyperoxidized form. Taken together, we conclude that PHA neurons are more sensitive to low doses of H2O2 than I2 neurons.

The difference in sensitivity between I2 and PHA neurons may come from distinct molecular mechanisms. To explore this possibility, we first tested which receptors are required in I2s and in PHAs for H2O2 perception. We focused on photoreceptors as photosensation is likely to involve the generation of ROS [8, 47]. In C. elegans, light sensing relies on unusual gustatory G-protein-coupled receptors (GPCRs) related to vertebrate photoreceptors: the two nematode closest paralogs LITE-1 and GUR-3 mediate photosensation in ASJ and ASH neurons [4749], and light and H2O2 sensing in I2 neurons [8]. We investigated whether these two receptors are differentially localized in I2 and PHA neurons. We generated a knock-in GUR-3::GFP line, which revealed that GUR-3 is solely expressed in I2 and I4 photosensory neurons (Fig 4A), as previously reported using episomal expression [8]. Such a restricted pattern deeply contrasts with the broad expression domain of LITE-1, which includes phasmid neurons (PHA and PHB), but not I2 neurons, as shown using both translational and transcriptional reporters [8].

Fig 4. H2O2 response involves different receptors and transducers in I2 and PHA neurons.

Fig 4

(A) Spinning-disc confocal projections of a representative GUR-3::GFP knock-in animal, at L1 (left) and L4 stages (right). The GUR-3::GFP signal is detected in I2 neurons and in a single I4 neurons (head panel), but not in PHA/PHB neurons (tail panel). Bar, 10μm. (B,D) Bar graph of the fraction of lite-1, gur-3, pmk-1 and sek-1 mutants responding to the H2O2 stimulation, classified as high (green), moderate (yellow) or absent (red) responses. N, number of movies analyzed. (C,E,F) Quantification of the calcium response to 1mM H2O2 in I2 and PHA neurons in controls and in gur-3 and lite-1 mutants (C), and in pmk-1 and sek-1 mutants at 1mM (E) and at 10μM H2O2 (F). Bars represent the mean (number of movies indicated) and error bars SD; ns, not significant, p>0.05; *p<0.05; ****p<0.0001 (ANOVA and Kruskal-Wallis tests). See corresponding S8S19 Movies and S7S10 Figs. (G,H) Blue light triggered different calcium fluxes in I2 and PHA neurons in the three regions analyzed; anterior neurite (green), posterior neurite (red) and soma (blue). The curves represent the mean and shaded error bars indicate SD. See S20S22 Movies.

These differential localizations prompted us to inquire whether mutants in these receptors were still able to trigger a response to 1mM H2O2 in I2 and PHA neurons. In gur-3(ok2245) mutants, only PHA neurons were able to respond to 1mM H2O2 (Fig 4B and 4C and S8 and S9 Movies and S7 and S8 Figs), providing evidence that GUR-3 function is not essential in PHA neurons for H2O2 sensing. In contrast, lite-1(ce314) mutants showed a reciprocal response, with only I2 neurons responding to 1mM H2O2 (Fig 4B and 4C and S10 and S11 Movies and S7 and S8 Figs). In conclusion, H2O2 likely activates I2 neurons via gur-3 and PHA neurons via its paralog lite-1, respectively. Interestingly, these observations might explain the previously reported impaired avoidance response of lite-1 mutants to 1mM H2O2 [8], as PHA neurons are involved in escape behavior [13].

PMK-1 and SEK-1 are required for PHA neurons response to micromolar H2O2 but are dispensable in I2 neurons

Since H2O2 perception in I2 and PHA neurons involves different receptors and requires the function of PRDX-2 in both cases, we wondered what type of signaling occurs downstream PRDX-2 to trigger neuronal activation upon H2O2 stimulation.

As mentioned above, studies in various models have reported that peroxiredoxins can modulate the p38/MAPK signaling pathway to influence cellular decisions, notably in drosophila and mammalian cells [34]. In C. elegans, specifically, the activation of this PRDX-2-PMK-1/p38MAPK cascade allows micromolar doses of H2O2 to potentiate the ASH neuron sensory behavior to glycerol [3]. Therefore, we investigated whether the p38/MAPK pathway could be involved in H2O2 sensing by analyzing the responses of pmk-1 (p38/MAPK) and sek-1 (MAPKK) mutants in I2 and PHA neurons. As the strongest allele pmk-1(ok811) is homozygous lethal, we had to use the hypomorphic pmk-1(km25) viable mutant, which carries a N-terminal deletion [50]. In pmk-1(km25) mutants, while I2 neurons responded normally to 1mM H2O2, PHA neurons showed a slightly milder response to 1mM H2O2 compared to controls (Fig 4D and 4E and S12 and S13 Movies and S9 Fig). In sek-1(km4) MAPKK mutants, we observed comparable responses of I2 and PHA neurons to 1mM H2O2 to that of controls (Fig 4D and 4E and S16 and S17 Movies and S10 Fig). We conclude that for 1mM H2O2 sensing, the p38/MAPK pathway is dispensable at least in I2 neurons, but may play some role in PHA neurons.

In ASH neurons, the PRDX-2-mediated activation of the p38/PMK-1 cascade induces a potentiation of their sensory behavior [3]. Therefore, we asked whether the p38/PMK-1 pathway could similarly promote PHA higher sensitivity to H2O2. We examined PHA neurons response to 10μM H2O2 in pmk-1 (MAPK) and in sek-1 (MAPKK) mutants. Strikingly, both mutants displayed a very similar phenotype, with an abolished response of PHA neurons to micromolar doses of H2O2 observed in pmk-1 (Fig 4D and 4F and S15 Movie and S9 Fig) and in sek-1 mutants (Fig 4D and 4F and S19 Movie and S10 Fig). In I2 neurons, as in controls, both pmk-1 and sek-1 failed to respond to 10μM H2O2 (Fig 4D and 4F and S14 and S18 Movies and S9 and S10 Figs), as expected. Taken together, this suggests that the p38/MAPK pathway would be specifically required for PHA neurons hypersensitivity to H2O2, but dispensable in I2 neurons.

PHA neurons are photosensory neurons like ASH neurons

Light sensing has been reported for ASJ, ASH and I2 neurons and require either the LITE-1 or the GUR-3 receptor, respectively [8, 4749]. As PHAs neurons require LITE-1 to respond to H2O2 (Fig 4B and 4C and S11 Movie and S7 Fig), we asked whether they could respond to light. To test this, we monitored calcium transients in three neuronal compartments using the GCaMP strain upon stimulating neurons with blue light, as previously done for I2 or ASH neurons [8, 47]. Interestingly, we found that all regions of PHA neurons responded to light, displaying a different response profile than those of I2 neurons (S22 Movie): PHA soma showed a stronger and longer response than I2 soma; PHA posterior neurite responded much slower that in I2 where it exhibits the fastest and strongest response peak, and the anterior neurite also had a slower recovery than in I2 (Fig 4G and 4H and S20 and S21 Movies). Overall, while I2 neurons exhibit a fast photoresponse within 10-15s, PHA neurons photoresponse requires twice longer to return to steady state (approx. 30s). Strikingly, we noticed that PHA neurons profile is highly reminiscent of that reported in ASH neurons photoresponse [47].

It has been proposed that light sensing may involve intracellular H2O2 release and peroxiredoxin signaling in both nematodes and yeast [7, 8]. To test whether PHA response to light requires the peroxiredoxin PRDX-2, we analyzed light sensing in prdx-2 mutants. As in H2O2 sensing (Fig 3E, 3F and 3H and S3 Movie), we observed that I2 response to light strictly depends on prdx-2 (15/15, S23 Movie and S11 Fig). Surprisingly, we found that PHA photoresponse does not require PRDX-2, as prdx-2 mutants were still able to respond to light (17/20, S24 Movie and S11 Fig). We conclude that light sensing does not involve the same mechanisms in I2 and in PHA neurons, downstream the photoreceptors.

Discussion

H2O2 sensing in head and tail neurons relies on different mechanisms

Here, we describe how two pairs of sensory neurons located in the head and the tail of C. elegans, namely I2s and PHAs, contribute to exogenous H2O2 and light sensing. Compared to previous reports, our study relies on a PRDX-2::GFP knock-in line more closely reflecting endogenous expression level, in comparison to overexpression often observed with extrachromosomal arrays. While classical methods do not enable precise control of the environment such as application of a stress, we carried out neuron response experiments using the microfluidic technology, allowing live imaging of immobilized animals upon simultaneous exposure to a controlled oxidative stress.

We found that PHA tail neurons can elicit a response to a micromolar range of H2O2, whereas I2 head neurons cannot, suggesting that distinct molecular mechanisms may account for this difference. Accordingly, while the peroxiredoxin PRDX-2 is essential for H2O2 sensing in both I2 and PHA neurons, a different transmembrane receptor is required to transduce the signal: I2 neurons use the gustatory receptor GUR-3, while PHA neurons would require its paralogue LITE-1. Another difference lies in the p38/MAPK activity requirement: while dispensable in I2 neurons, it would be specifically required in PHA neurons to confer their hypersensitivity to H2O2 (lack of response of PHA neurons to 10μM H2O2 in sek-1/MAPKK and in pmk-1/MAPK mutants, Fig 4). Finally, although nematodes were known for a long time to avoid light when the light pulse was applied on their tail [48], our work provide the first evidence, to our knowledge, that PHA tail neurons act as photoreceptor cells.

Overall, our data are consistent with previous findings unveiling the existence of two distinct modes of response to oxidative stress in C. elegans: a direct response in peripheral tissues such as the gut, and a neuronally-regulated response relying on synaptic transmission [51]. Specifically, we uncovered that a harsh oxidative stress (10mM H2O2) triggers PRDX-2 induction in the anterior gut and in the EPC (Figs 1 and 2), while lower doses of H2O2 triggers either I2 and PHA neurons activation (1mM), or only PHA neurons activation (10μM) (Fig 3). Interestingly, both types of response involve the peroxiredoxin PRDX-2, which behaves differently in the two cellular contexts: PRDX-2 would be cell-autonomously induced by SKN-1 in the intestine, but likely not in neurons (Fig 2). Therefore, we propose that PRDX-2 might act as a peroxidase in the gut, as proposed [27], whereas it could function as a H2O2-signaling molecule in neurons, as suggested for I2 neurons [8]. Importantly, H2O2 response in I2 and PHA neurons requires the joint function of PRDX-2 and a receptor, as each mutant individually cannot respond (Figs 3 and 4). In addition, former rescue experiments indicated that light response requires the activity of PRDX-2 and GUR-3 specifically in I2 neurons [8]. Based on all these data and recent studies shedding light on how H2O2 is sensed in plant and animal cells [34, 52], we propose new hypotheses regarding H2O2 signaling in neurons which are depicted in Fig 5 and described below, to integrate our findings with recent results from the literature.

Fig 5. Hypothetical model of H2O2 sensing and signaling in C. elegans I2 and PHA neurons based on this and on previous work.

Fig 5

(A-B) Hypothetical model of H2O2-induced neuronal activation, based on our data and on previous studies. Schematic drawing of a presynaptic button in I2 (A) and PHA (B) neurons, illustrating the presumptive H2O2-PRDX-2-mediated neuronal activation in both cases (modelled in S12 Fig). High doses of H2O2 (1mM) are sensed by both neurons, but only PHA neurons respond to 10μM H2O2, as illustrated in red. In this model, H2O2 would freely diffuse through the neuron plasma membrane and oxidize PRDX-2, presumably leading to LITE-1 or GUR-3 activation. Receptor activation is likely relayed by cGMP signaling, resulting in the opening of voltage-gated calcium channels (in grey) and neurotransmitter release (glutamate in both cases), triggering an adapted response. In PHA neurons, the PMK-1/p38MAPK pathway is additionally required to promote neuronal response to micromolar doses of H2O2, potentially through OSM-9 phosphorylation, as observed in ASH neurons [3]. See Discussion for further details and bibliographic references.

A presumptive model of H2O2 sensing in C. elegans neurons

In both I2 and PHA neurons, we favor the hypothesis that cytosolic PRDX-2 rather than the transmembrane receptor would be the neuronal H2O2 sensor, based on the following observations: i) in many cases, H2O2 signaling is mediated by oxidation of cysteines in redox-regulated proteins [53]. Alternatively, redox signaling often relies on peroxiredoxins acting as sensor and transducer of H2O2 signal [54], as thiol modifications would be much faster when catalyzed by peroxiredoxins [5557], due to their abundance and their high reactivity to H2O2 [58]. Here, the striking abundance of PRDX-2 in PHA, and especially in I2 neurons reinforces this idea, as well as the defective response of prdx-2 mutants to both doses of H2O2 tested (Figs 3 and S6). Consequently, we propose that H2O2 signaling in I2 and PHA neurons would involve redox signaling through PRDX-2. ii) Concerning receptor topology, GUR-3 and LITE-1 present a higher conservation in their intracellular domains, with conserved cysteines only found intracellularly and in a transmembrane domain (S12A Fig). This structure is more reminiscent of that of the vertebrate transmembrane protein GDE2, which is activated intracellularly by the Prdx1 peroxiredoxin [59], than of the plant H2O2 sensor, the HCPA1 receptor, whose activation involves direct oxidation of extracellular cysteines [52]. Here, GUR-3 and LITE-1 receptor topology does not support the hypothesis of a direct oxidation by H2O2 on the extracellular domain, but rather suggests intracellular signaling as in the case of GDE2 (S12B and S12C Fig). We thus propose a scenario in which H2O2 would diffuse through the neuron plasma membrane and oxidize PRDX-2. Oxidized PRDX-2 or its disulfide form (PRDX-2ox/S-S) would in turn react with the cysteines of GUR-3 or LITE-1, triggering receptor activation, and I2 or PHA neuron response (Figs 5 and S12). These hypotheses will require future experimental validation.

Neuronal response involves the opening of calcium channels, which are different in I2 and PHA neurons: I2 activation depends on UNC-2 and UNC-36 voltage-gated calcium channels [9], while PHA neurons require the cyclic nucleotide-gated channel TAX-4 and the vertebrate TRPV5 nematode equivalent OSM-9 [12]. Of note, the other TRPV5 channel subunit OCR-2 may also act in PHAs, as it is expressed in PHAs (S13 Fig, [60]) and it functions together with OSM-9 [61]. In conclusion, although closely related and both able to sense H2O2 and light, GUR-3 and LITE-1 receptor signaling likely involves different downstream transducers. Except for PRDX-2 requirement, I2 and PHA neurons would use distinct molecular pathways to transduce H2O2 response.

A striking parallel between PHA and ASH neurons

Our data uncover a high sensitivity of PHA neurons to micromolar doses of H2O2 that is not seen in I2 neurons and requires PRDX-2 and p38/MAPK activity. Strikingly, micromolar doses of H2O2 were also reported to activate the p38/MAPK pathway via PRDX-2 in ASH neurons, leading to the phosphorylation of the OSM-9 TRPV sensory channel, thereby increasing its sensitivity [3]. Although our study and the latter do not elucidate how PRDX-2 triggers PMK-1 activation, recent evidence sheds light on this process: in both mammalian and drosophila cells, H2O2 induces transient disulfide-linked conjugates between the MAP3K and a typical 2-Cys peroxiredoxin [34]. Similarly, in C. elegans, PRDX-2 could activate the MAPKKK NSY-1, as NSY-1 function is required in ASH neuron [3], and may be expressed in PHAs (unidentified cells of the tail, D. Moerman, WormBase). Based on these observations and on the fact that OSM-9 is expressed [62] and likely required in PHA neurons [12], it is possible that PMK-1/p38MAPK activation might increase PHA neurons’ sensitivity to H2O2 through the downstream phosphorylation of OSM-9, via AKT-1, as in ASH neurons (Fig 5). Alternatively, by analogy with recent work showing that DLK/p38MAPK signaling controls LITE-1 stability in ASH neurons, PMK-1 could also control LITE-1 turnover by RAB-5-mediated endocytosis [63]. These assumptions, which will necessitate further experimental testing, are supported by the observation that ASHL/R and PHAL/R are both descendants of ABplp or ABprp in the nematode cell lineage, and hence may express similar sets of genes following lineage-specific priming [64].

The fact that PHA, PHB and ASH were found in the same neuron cluster [65] confirms that they share similar molecular signatures. To illustrate the parallel between ASH and PHA polymodal nociceptors, we retrieved the set of genes expressed in these neurons [65], and analysed which ones were specifically enriched in these neurons in comparison to all other neurons (S13 Fig, data in S1S3 Tables). This analysis revealed that ASH and PHA not only express many genes specific to ciliated neurons, as expected (eg. che-3, nphp-4, che-11, ifta-1, C33A12.4 and R102.2, [66]), but also a number of common receptors (eg. ocr-2, osm-9, ida-1, casy-1, ptp-3 or pdfr-1), although some of them are more enriched in ASH (dop-2, nrx-1, snt-5, sue-1). Intriguingly, their neuropeptide profile appears different for the two neuron pairs (PHA enriched in flp-7, flp-4, flp-16, nlp-1 ins-18, while ASH strongly express flp-13 and npr genes), suggesting that beyond their functional similarity, ASH and PHA may trigger different types of intercellular communication. Finally, this analysis highlights the fact that several genes with unknown function are highly enriched in both ASH and PHA (eg. F27C1.11, W05F2.7, tos-1, cab-1), pointing to their potential role in neuronal function.

Taken together, our analyses highlight the common features shared between PHA and ASH polymodal nociceptive neurons, as formerly noticed [13], since they both: i) display a higher sensitivity dependent on the p38/MAPK pathway (Fig 4D–4F and [3]), ii) exhibit a similar response profile to light (Fig 4H and [47]), iii) require the photoreceptor LITE-1 for lightsensing [47] or H2O2 sensing (Fig 4B and 4C), iv) trigger avoidance [13], and v) share a close molecular signature (S13 Fig and [65]). A recent report indicates that ASJ neurons are also required for H2O2 avoidance [6], and unravels the behavioral mechanisms allowing C. elegans to find a suitable niche, owing to the interplay between H2O2 and bacteria in its environment. Overall, these observations led us to propose that nematodes might integrate the environmental redox signals from at least four different pairs of neurons (I2, ASH, PHA and ASJ) in order to trigger an appropriate dose-dependent physiological response.

GUR-3 and LITE-1 receptors mediate both light and H2O2 sensing

In yeast, light sensing relies on the peroxisomal oxidase Pox1 which triggers light-dependent H2O2 formation, the latter being sensed by the Tsa1 peroxiredoxin and transduced to thioredoxin for subsequent signaling [7]. Nematodes, unlike yeast, require a photoreceptor in addition to the antioxidant for lightsensation: GUR-3 in I2 neurons [8], and LITE-1 in ASJ and ASH neurons [47, 49]. Here we showed that both I2 and PHA respond to light, but with a different profile. Despite its unusual membrane topology, LITE-1 has been shown to encode a bona fide photoreceptor, whose photoabsorption depends on its conformation [67]. However, whether light directly activate the neuron photoreceptor or triggers intracellular H2O2 release and signaling is still unclear. In agreement with the fact that H2O2 inhibits LITE-1 photoabsorption in vitro [67], it has been shown that a H2O2 pretreatment reduces LITE-1-mediated photoresponse in ASH neurons [47]. Consistent with these reports, our data illustrate that GUR-3 and LITE-1 have a dual function in both light and H2O2 sensing. In I2 neurons, as in yeast, redox signaling could be involved in transducing the light signal, as PRDX-2 is strictly required for light sensing (S11 Fig, [8]). In contrast, PHA neurons can respond to light without PRDX-2, indicating that the LITE-1 photoreceptor tranduces the light signal without a redox relay. This difference could explain our observation that I2 neurons respond faster to light than PHA and ASH neurons (Fig 4G and 4H and [47]). Whether this difference strictly depends on the photoreceptor (LITE-1 in PHA, ASH vs GUR-3 in I2) and/or on its downstream signaling cascade is an open question.

Finally, it is noteworthy that I2 and PHA neurons rely on peroxiredoxin for H2O2 and/or light sensing, while ASJ and ASH rely on thioredoxin: TRX-1 is required for LITE-1-dependent photosensation in ASH neurons [47], and is expressed in ASJ photosensory neurons [68], which respond to H2O2 [6]. Altogether, this further underlines the importance of a redox signaling relay involving antioxidants of the peroxiredoxic cycle in nematode photosensory neurons.

In conclusion, our work illustrates that nematodes can sense various concentrations of H2O2 through sensory neurons located in the head and the tail, using either partially different (I2 and PHA), or similar molecular mechanisms (ASH and PHA). This set of neurons also confer light sensing to the nematode with a distinct speed in the response (fast in I2, slow in PHA and ASH, [47]). While I2 neurons seem more specialized in sensing oxidative stress [8, 10], PHA and ASH can detect many other stimuli [12], but all of them can trigger avoidance. To tackle the complex question of how these neuronal inputs translates into behavior, a recent method was developed [69], allowing the simultaneous recording of behavioral and neural responses of C. elegans to salt concentrations changes. These observations may help uncover whether and how inputs from head and tail oxidative stress sensory neurons integrate to allow nematodes to quickly and appropriately react to a change in the environment.

Experimental procedures

Generation of plasmids and transgenic strains by CRISPR/Cas9-mediated genome editing

C. elegans strains (listed in Supplementary Information) were maintained as described [70]. PRDX-2::GFP knock-in strain was generated by CRISPR/Cas9-mediated genome editing, using a DNA plasmid-based repair template strategy [37]. For both PRDX-2 and GUR-3 knock-ins, a C-terminal GFP fusion was generated, comprising a flexible linker between the coding region and GFP to allow correct folding of the fusion protein. A combined small guide-RNA/repair template plasmid was built using the SAP Trap strategy [71]. Phusion DNA polymerase was used to amplify by PCR 5’ and 3’ prdx-2 and gur-3 homology arms (HAs) from N2 genomic DNA, using primers containing SapI restriction sites and silent mutations to prevent Cas9 re-cleavage (all primers sequences listed in Supplementary Information). After purification, 5’ and 3’ HAs and sgRNA oligonucleotides were assembled into the destination vector (pMLS256), together with the flexible linker (from pMLS287), GFP and the unc-119 rescuing element (from pMLS252), in a single SapI restriction-ligation reaction, as described [71]. Prior to transformation in DH5α cells, a sabotage restriction was performed with SpeI to digest empty destination vectors but not the desired assembly constructs, which were subsequentially verified by restriction digest analysis and sequencing. All plasmids used for injection were purified using a DNA Miniprep Kit (PureLink, Invitrogen), or a DNA midiprep kit (Macherey Nagel). For PRDX-2::GFP knock-in, a plasmid mix containing combined sgRNA/repair template plasmid (50 ng/μl), Cas9-encoding pSJ858 (25ng/μl) and co-injection markers (pCFJ90 at 2.5ng/μl; pCFJ104 at 5ng/μl, and pGH8 at 5ng/μl) was injected in the germline of unc-119(ed3) animals [37]. For GUR-3::GFP knock-in, an injection mix containing purified Cas9 protein (IDT) associated with tracrRNA and crRNA (guide RNA), GUR-3 repair template, and co-injection markers was injected in unc-119(ed3) animals, according to IDT online protocols for C. elegans. Plates containing 2/3 injected F0 animals were starved, chunked on fresh plates, for candidates screening (attested by the presence of wild-type non fluorescent animals). Knock-in events were validated by PCR on homozygous lysed worms (QuantaBio AccuStart II GelTrack PCR SuperMix), using primers annealing in the inserted sequence and in an adjacent genomic region not included in the repair template. The PRDX-2::GFP strain was outcrossed 5 times to N2 wild-types.

RNA interference

RNAi experiments were performed by feeding using the Ahringer-MRC feeding library [72]. Animals fed with the empty vector L4440 served as a negative control. The efficiency of each RNAi experiment was assessed by adding an internal positive control, zyg-9(RNAi), which induces embryonic lethality.

Spinning-disk confocal microscopy acquisitions and fluorescence intensity measurements

For live imaging, animals were anesthetized in M9 containing 1mM levamisole and mounted between slide and coverslip on 3% agarose pads. Synchronized L4 animals were treated for 30min in a 96-well flat bottom plate, in 50μl of M9 containing 1mM or 10mM H2O2. Treated animals were transferred using a siliconized tip on a freshly seeded plate to recover, and imaged 1h30 to 2h later. Spinning-disk confocal imaging was performed on a system composed of an inverted DMI8 Leica microscope, a Yokogawa CSUW1 head, an Orca Flash 4.0 camera (2048*2018 pixels) piloted by the Metamorph software. Objective used were oil-immersion 40X (HC PL APO, NA 1.3) or 63X (HCX PL APO Lambda blue, NA 1.4). The temperature of the microscopy room was maintained at 20˚C for all experiments. Z-stacks of various body regions were acquired with a constant exposure time and a constant laser power in all experiments. Maximum intensity projections were used to generate the images shown. Fluorescence intensity measurements in int1, I2 and EPC cells were performed using the Fiji software, by manually drawing a region of interest (ROI) around the cell (int1, EPC), or applying a threshold (I2 neurons), background was subtracted and average pixel intensity was quantified.

Microfabrication and microfluidic chip preparation

The microfluidic chip original design was inspired by the Wormspa [73], but pillars distances were adapted to trap L4 animals, and multiple series of traps were included to increase the number of experiments per chip (S4 Fig). A master mold was made by standard soft photolithography processes by spin-coating a 25μm layer of SU-8 2025 (Microchem, USA) photoresist at 2700 rpm for 30sec on a 3" wafer (Neyco, FRANCE). Then, we used a soft bake f 7min at 95°C on hot plates (VWR) followed by a UV 365nm exposure at 160 mJ/cm² with a mask aligner (UV-KUB3 Kloé®, FRANCE). Finally, a post-exposure baking identical to the soft bake was performed before development with SU-8 developer (Microchem, USA). Then, the wafer was baked at 150°C for 15min to anneal potential cracks and strengthen the adhesion of the resist to the wafer. Finally, the master mold was treated with chlorotrimethylsilane to passivate the surface.

Worm microchannels were cast by curing PDMS (Sylgard 184,10:1 mixing ratio), covalently bound to a 24 × 50 mm coverslip after plasma surface activation (Diener, Germany), and incubated 20min at 60°C for optimal adhesion. The chip was perfused with filtrated M9 solution through the medium inlet using a peristaltic pump (Ismatec), until complete removal of air bubbles. Worm loading was performed with the pump set at a low flow rate (<30 μl/min), through a distinct inlet (S4 Fig): 10–15 young L4 animals (synchronized by bleaching 48h prior to each experiment) were picked in a siliconized Eppendorf tube (Sigmacote SL2, Sigma Aldrich) containing M9, and perfused into the traps. The loaded chip was carried to the microscope while being still connected to the pump by a gravity flow (preventing animals to escape) until the microfluidic chip was installed on the microscope stage.

Calcium imaging

I2 and PHA neuronal response was monitored using the calcium sensor GCaMP3 expressed under the flp-15 promoter as in [8]. To image H2O2 response, young L4 animals trapped in microfluidic chips were imaged using the confocal spinning disc system described above with the 20X air objective (HC PL APO CS2, NA 0.75). The microfluidic chip allowed the simultaneous recording of up to 3 animals per experiment (S4 Fig). Z-stacks of 10–15 images (10 μm spacing) were acquired every 2s (using the stream Z mode), for 350 time points. Exposure time was 50ms and laser power set on 40%. The device was perfused with M9 medium throughout the experiment using a peristaltic pump set at 80 μl/min, and H2O2 was perfused (at 10μM or 1mM in M9) for 100 time points (3min20s) after an initial recording of 35–45 time points. Movies were computationally projected using MetaMorph, and data processing (including movie registration, neuron segmentation and tracking over time) was conducted with a custom-developed Matlab program detailed below and available at https://github.com/gcharvin/viewworm (a user guide is provided in Supplementary Information).

To image light response in I2 and PHA neurons, L4 worms were mounted on a slide covered with 3% agarose pads in M9 supplemented with 1mM levamisole. Video-recordings were performed on the spinning-disc microscope using the 40X oil objective. Animals were exposed to blue light (485nm) while their neuronal response was simultaneously recorded in stream mode (10 frames/sec, single Z, 100ms exposure, laser 100%) for 30sec.

Calcium response analyses

For H2O2 response analyses, sequences of images were spatially realigned with respect to the first image of the timeseries in order to limit the apparent motion of the worm in the trap and ease the tracking of neurons of interest. This image registration process was performed using standard 2D image cross-correlation by taking the first image as a reference. Then, we used a machine-learning algorithm (based on a decision tree) to segment pixels in the fluorescent images. For this, we took a series of image transforms (gaussian, median, range filters) as descriptors for the classifier, and we trained the model on typically 10 frames before applying the result to the rest of the time series. This segmentation method appeared to be superior to simple image thresholding, which is inadequate when dealing with fluorescent signals that vary both in time and space (the brightness of two neurons is quite different). Next to the segmentation procedure, we tracked the identified neurons using distance minimization, and we quantified the mean fluorescence signal in each neuron over time. Last, fluorescence data corresponding to individual animals were pooled after synchronization from the time of exposure to H2O2 and signal normalization. This image analysis pipeline is available at https://github.com/gcharvin/viewworm and a tutorial for use is included in Supplementary Information. As some movies could not be quantified due to uncontrolled animal movements, a visual classification of neuronal responses (high, moderate, absent) was made by comparison to successfully tracked movies.

For light response analyses in I2 and PHA neurons, the same image processing pipeline as in [8] was used, except that ROI were manually drawn in Fiji.

Statistical analyses

For pairwise comparisons of data sets with a normal distribution (or N>30), p-values were calculated using an unpaired two-tailed Student test, and the Welch correction was applied when samples variance was not homogeneous. When distributions were not normal (Anderson-Darling and Shapiro Wilk tests not satisfied), a Mann Whitney test was used. Multiple comparisons were analyzed using a one-way ANOVA with Bonferroni’s correction (for normal distributions), or a non-parametrical Kruskal-Wallis test followed by a Dunn’s multiple comparisons test. Statistical analyses were conducted with the GraphPad Prism9 software.

Mean are represented and error bars indicate standard deviation (SD) in all figures. The data presented here come from at least three independent experiments. For p values, not significant p>0.05; *p<0.05; **p<0.01; ***p<0.001: ****p<0.0001.

Supporting information

S1 Fig. Related to Fig 1- The available PRDX-2::GFP transgenic line (CTD1051.3) does not suit to our study.

(A) Low magnification fluorescent image of untreated animals from the CTD1051.3 line showing that transgenics express aggregates of PRDX-2::GFP, a hallmark of overexpression of the fusion protein. (B,C) Low magnification images of animals treated in flat bottom wells imaged after 24h of treatment in the potent oxidative stress inducing agent tBOOH. In the wild-type control well (A), almost all animals are all dead in 2mM tBOOH, appearing as rods, while CTD1051.3 transgenics survive a 25X higher dose of the drug, indicating their much stronger resistance to oxidative stress. (D) Representative image of an animal of the PRDX-2::GFP knock-in line; a wild-type control imaged with the same settings is shown (E), delineated by a dotted line. Bar, 100μm. (F-H) Confocal projections showing the tail region (dotted contours) of a PRDX-2::GFP transgenic animal stained with the lipophilic orange-red dye DiI (see https://www.wormatlas.org/EMmethods/DiIDiO.htm), imaged in green (F) and red channels (G). The overlay (H) shows that PRDX-2::GFP tail neurons are stained by the dye, establishing their identity as phasmid sensory neurons (PHA/PHB). Bar, 10μm.

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S2 Fig. Related to Figs 1 and 2- PRDX-2 induction is not observed in I2 neurons upon H2O2 treatment.

(A,B) Wild-type controls do not show a higher gut autofluorescence after H2O2 treatment, as illustrated by the int1 anterior gut cell fluorescence quantification (C). (D,E) Quantification of the PRDX-2::GFP fluorescence level in I2 neurons and in the excretory pore cell (EPC) in controls after 1mM-H2O2 treatment. (F,G) Quantification of I2 neurons’ PRDX-2::GFP fluorescence level in skn-1(zj15) mutants and in skn-1(RNAi) animals upon a 10mM-H2O2 treatment. Means are shown and error bars represent SD; ns, not significant, p>0.05 (t test or Mann-Whitney test). Scale bar, 50μm.

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S3 Fig. Related to Fig 2.

Genomic organization of the prdx-2 locus and presumptive regulation by SKN-1 and additional transcription factors. (A) Genome Browser screenshot from WormBase (release WS283, JBrowse II), showing the gene organization on chromosome II, at the indicated coordinates (top), and the peaks detected by ChIP-seq using an anti-GFP antibody [42] in prdx-2 and pkc-3 promoters, in GFP-tagged transgenic lines of the indicated transcription factors (B). Note the co-regulation of prdx-2 and pkc-3, which are organized in an operon, illustrated by the green bar.

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S4 Fig. Related to Figs 3 and 4- Design of the microfluidic chip used in H2O2 neuron response experiments.

(A) Global view of the microfluidic chip used in all neuron response experiments, showing the 6 independent series of 10 worm traps. The original file (Autocad format) is available at https://github.com/gcharvin/viewworm. (B) Magnification of the worm trap area showing the 10 individual channels (corresponding to the red box in A). Scale bar, 1mm. (C) DIC image of an animal trapped (anterior to the left). Occasionally animals were trapped in the opposite orientation, but this did not affect the neuronal response. Scale bar, 100μm.

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S5 Fig. Related to Fig 3- Individual intensity measurements of S1S7 Movies.

The curves represent the mean GCaMP3 intensity raw value over time (1 frame = 2sec) quantified in I2 and PHA neurons in control and prdx-2 mutants corresponding movies (S1S7 Movies), upon a 1mM or a 10μM H2O2 exposure. T0 indicate the time point at which the H2O2 treatment has been applied during 100 frames. Red and blue colors represent left and right I2 and PHA neurons (ie I2L/R and PHAL/R). Note that the colors have been changed in normalized average curves shown in Fig 3 and in related S6 Fig.

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S6 Fig. Related to Fig 3- prdx-2 mutants PHA neurons do not respond to 10μM H2O2.

(A-D) Average curves showing the normalized calcium response to 10μM H2O2 measured over time (in seconds) using the GCaMP3 sensor in PHA left and right neurons (top and bottom curves) in prdx-2(gk169) mutants (B) and in wild-type controls (A). N, number of movies analyzed for each genotype. See S6S7 Movies and related S5 Fig.

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S7 Fig. Related to Fig 4- gur-3 and lite-1 mutants show reciprocal phenotypes in H2O2 sensing in I2 and in PHA neurons.

(A-D) Average curves showing the normalized calcium response to 1mM H2O2 measured over time (indicated in seconds) using the GCaMP3 sensor in I2 and PHA left and right neurons (top and bottom curves) in gur-3(ok2245) (A,B) and lite-1(ce314) mutants (C,D). N, number of movies analyzed for each genotype. See S8S11 Movies and related S8 Fig.

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S8 Fig. Related to Fig 4- Individual intensity measurements of S8S11 Movies (gur-3 and lite-1 mutants).

The curves represent the mean GCaMP3 intensity raw value over time (1 frame = 2sec) quantified in I2 and PHA neurons in gur-3 and lite-1 mutants upon a 1mM H2O2 exposure (starting at T0 and lasting 100 frames), in corresponding movies (S8S11 Movies). Red and blue colors show left and right neurons for I2 (left panel) and PHA (right panel) neurons (ie I2L/R and PHAL/R). Note the reciprocal phenotypes observed in both mutants. Colors have been changed in the related normalized average curves shown in S7 Fig.

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S9 Fig. Related to Fig 4, continued- Individual intensity measurements of S12S15 Movies (pmk-1 mutants).

The curves represent the mean GCaMP3 intensity raw value over time (1 frame = 2sec) quantified in I2 and PHA neurons in pmk-1 mutants upon a 1mM or a 10μM H2O2 stimulation (starting at T0 and lasting 100 frames), in corresponding movies (S12S15 Movies). Red and blue color indicate I2L/R neurons (left panel) and PHAL/R neurons (right panel).

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S10 Fig. Related to Fig 4, continued- Individual intensity measurements of S16S19 Movies (sek-1 mutants).

The curves represent the mean GCaMP3 intensity raw value over time (1 frame = 2sec) quantified in I2 and PHA neurons in sek-1 mutants upon a 1mM or a 10μM H2O2 stimulation (starting at T0 and lasting 100 frames), in corresponding movies (S16S19 Movies). Red and blue color indicate left and right I2 (left panel) and PHA (right panel) neurons.

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S11 Fig. Related to Fig 4, continued—Unlike in I2 neurons, PRDX-2 is not essential for light sensing in PHA neurons.

Average curves showing the normalized calcium response to blue light over time (in seconds) using the GCaMP3 sensor, measured in the soma of I2 (A) and PHA neurons (B) in wild-type controls and in prdx-2(gk169) mutants. N, number of movies analyzed for each genotype. Error bars represent SD. While I2 neurons fail to respond to light in prdx-2 mutants, PHA neurons do respond, albeit with a lower intensity peak than in controls. See related S20S24 Movies.

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S12 Fig. Related to Discussion- A presumptive model of how GUR-3 and LITE-1 receptors may be activated intracellularly by PRDX-2.

(A) Alignment of LITE-1 and GUR-3 protein sequences made with the SIM alignment tool (https://web.expasy.org/sim/), using the comparison matrix BLOSUM30. Transmembrane (TM) and intra/extracellular domains were predicted using the DeepTMHMM program (https://dtu.biolib.com/DeepTMHMM). The alignment reveals 39.9% identity over 434 residues overlap. Note the rather large intracellular domains (in blue), encompassing conserved cysteines (boxed in red) (B-C) A putative PRDX-2 redox relay may trigger H2O2-induced receptor activation in I2 and PHA neurons. Sketch of GUR-3 (B) and LITE-1 (C) receptors, deduced from A, depicting their conserved cysteines. Upon H2O2 exposure, oxidized PRDX-2 or its disulfide form (PRDX-2ox/S-S) could oxidize these cysteines, possibly forming a disulfide conjugate and/or inducing a conformation change, which would subsequently trigger receptor activation, and I2 or PHA neuron response.

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S13 Fig. Related to Discussion- PHA and ASH neurons belong to the same neuronal cluster, and share partially overlapping transcriptomic signatures.

(A) Uniform Manifold Approximation and Projection (UMAP) projection of parent cluster 28, from [65] in which ASH and PHA/PHB nociceptive neurons were found in distinct sub-clusters (Louvain clustering at 8 PCs and at 1.2 resolution). (B, C) Dot plot indicating for a selection of genes both the intensity of gene expression and the fraction of expressing cells in each sub-cluster (B), based on single-cell RNA-sequencing data from [38]. See Supplementary Information and related S1S3 Tables for exhaustive lists of genes expressed in each cluster.

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S14 Fig

(TIF)

S1 Movie. (Related to Fig 3)- Wild-type I2 response to 1mM H2O2.

On-chip 4D movie showing control I2 neurons response to 1mM H2O2 visualized using the GCaMP3 calcium sensor. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S5 Fig.

(MP4)

S2 Movie. (Related to Fig 3)- Wild-type PHA response to 1mM H2O2.

On-chip 4D movie showing control PHA neurons response to 1mM H2O2 visualized using the GCaMP3 calcium sensor. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S5 Fig.

(MP4)

S3 Movie. (Related to Fig 3)- prdx-2 mutant I2 response to 1mM H2O2.

On-chip 4D movie showing the absence of I2 neurons response to 1mM H2O2 in a prdx-2 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S5 Fig.

(MP4)

S4 Movie. (Related to Fig 3)- prdx-2 mutant PHA response to 1mM H2O2.

On-chip 4D movie showing the absence of PHA neurons response to 1mM H2O2 in a prdx-2 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S5 Fig.

(MP4)

S5 Movie. (Related to Fig 3)- Wild-type I2 response to 10μM H2O2.

On-chip 4D movie showing the absence of I2 neurons response to 10μM H2O2 in a control animal. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S5 Fig.

(MP4)

S6 Movie. (Related to Fig 3)- Wild-type PHA response to 10μM H2O2.

On-chip 4D movie showing control PHA neurons response to 10μM H2O2 visualized using the GCaMP3 calcium sensor. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S5 Fig.

(MP4)

S7 Movie. (Related to S6 Fig)- prdx-2 mutant PHA response to 10μM H2O2.

On-chip 4D movie showing the absence of PHA neurons response to 10μM H2O2 in a prdx-2 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S5 Fig.

(MP4)

S8 Movie. (Related to Fig 4)- gur-3 mutant I2 response to 1mM H2O2.

On-chip 4D movie showing the absence of I2 neurons response to 1mM H2O2 in a gur-3 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S8 Fig and related average response in S7 Fig.

(MP4)

S9 Movie. (Related to Fig 4)- gur-3 mutant PHA response to 1mM H2O2.

On-chip 4D movie showing PHA neurons response to 1mM H2O2 in a gur-3 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S8 Fig and related average response in S7 Fig.

(MP4)

S10 Movie. (Related to Fig 4)- lite-1 mutant I2 response to 1mM H2O2.

On-chip 4D movie showing I2 neurons response to 1mM H2O2 in a lite-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S8 Fig and related average response in S7 Fig.

(MP4)

S11 Movie. (Related to Fig 4)- lite-1 mutant PHA response to 1mM H2O2.

On-chip 4D movie showing the absence of PHA neurons response to 1mM H2O2 in a lite-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S8 Fig and related average response in S7 Fig.

(MP4)

S12 Movie. (Related to Fig 4)- pmk-1 mutant I2 response to 1mM H2O2.

On-chip 4D movie showing I2 neurons response to 1mM H2O2 in a pmk-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S9 Fig.

(MP4)

S13 Movie. (Related to Fig 4)- pmk-1 mutant PHA response to 1mM H2O2.

On-chip 4D movie showing PHA neurons response to 1mM H2O2 in a pmk-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S9 Fig.

(MP4)

S14 Movie. (Related to Fig 4)- pmk-1 mutant I2 response to 10μM H2O2.

On-chip 4D movie showing the absence of I2 neurons response to 10μM H2O2 in a pmk-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S9 Fig.

(MP4)

S15 Movie. (Related to Fig 4)- pmk-1 mutant PHA response to 10μM H2O2.

On-chip 4D movie showing the absence of PHA neurons response to 10μM H2O2 in a pmk-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S9 Fig.

(MP4)

S16 Movie. (Related to Fig 4)- sek-1 mutant I2 response to 1mM H2O2.

On-chip 4D movie showing I2 neurons response to 1mM H2O2 in a sek-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S10 Fig.

(MP4)

S17 Movie. (Related to Fig 4)- sek-1 mutant PHA response to 1mM H2O2.

On-chip 4D movie showing PHA neurons response to 1mM H2O2 in a sek-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S10 Fig.

(MP4)

S18 Movie. (Related to Fig 4)- sek-1 mutant I2 response to 10μM H2O2.

On-chip 4D movie showing the absence of I2 neurons response to 10μM H2O2 in a sek-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S10 Fig.

(MP4)

S19 Movie. (Related to Fig 4)- sek-1 mutant PHA response to 10μM H2O2.

On-chip 4D movie showing the absence of PHA neurons response to 10μM H2O2 in a sek-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S10 Fig.

(MP4)

S20 Movie. (Related to Fig 4)- Wild-type control I2 response to light.

Single-Z movie (recorded at 10 fps) showing control I2 neurons response to blue light (485nm), visualized with the GCaMP3 calcium sensor. The original movie has been colored artificially to highlight fluorescence intensity variations, using Fiji ‘glow’ look up table. Note the quasi instantaneous and very strong posterior neurite response. Accelerated 5 times.

(AVI)

S21 Movie. (Related to Fig 4)- Wild-type control PHA response to light.

Single-Z movie (recorded at 10 fps) showing control PHA neurons response to blue light (485nm), visualized with the GCaMP3 calcium sensor. The original movies has been colored artificially to highlight fluorescence intensity variations, using Fiji ‘glow’ look up table. Note the long-lasting PHA soma response. Accelerated 5 times.

(AVI)

S22 Movie. (Related to Fig 4)- Wild-type control I2 and PHA simultaneous responses to light.

Single-Z movie (recorded at 10 fps) showing simultaneously control I2 (right) and PHA (left) neurons response to blue light (485nm). The original movies has been colored artificially to highlight fluorescence intensity variations, using Fiji ‘glow’ look up table. Note the contrast of response, with the quasi instantaneous response of I2 neurons versus the slower and longer soma response in PHA neurons. Accelerated 5 times.

(AVI)

S23 Movie. (Related to Fig 4)- prdx-2 mutant I2 response to light.

Single-Z movie (recorded at 10 fps) showing the absence of I2 neurons response to blue light (485nm) in a prdx-2 mutant, visualized with the GCaMP3 calcium sensor. The original movie has been colored artificially to highlight fluorescence intensity variations, using Fiji ‘glow’ look up table. Accelerated 5 times. See related average response in S11 Fig.

(AVI)

S24 Movie. (Related to Fig 4)- prdx-2 mutant PHA response to light.

Single-Z movie (recorded at 10 fps) showing PHA neurons response to blue light (485nm) in a prdx-2 mutant, visualized with the GCaMP3 calcium sensor. The original movie has been colored artificially to highlight fluorescence intensity variations, using Fiji ‘glow’ look up table. Note the long-lasting PHA soma response in prdx-2 mutant as in WT (S21 Movie). Accelerated 5 times. See related average response in S11 Fig.

(AVI)

S1 Graphical abstract

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S1 File

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S2 File

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S1 Table

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S2 Table

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S3 Table

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Acknowledgments

We are grateful to all the staff members of the Imaging Center of the IGBMC, especially Elvire Guiot, Marine Silvin, Erwan Grandgirard and Bertrand Vernay for assistance in confocal microscopy. We thank Christelle Gally, Basile Jacquel and Eric Marois for helpful discussions and critical reading of the manuscript, Sandra Bour for assistance with figure design, Doulaye Dembele for help with statistic analyses. We are indebted to the Reymann, Vermot and Jarriault labs for sharing their equipments and reagents, and for scientific input. We thank the Horvitz lab, especially Na An, for providing the MT GCaMP strains and the Pujol lab for providing the sek-1 mutant. We thank WormBase for providing resources and the Caenorhabditis Genetics Center (funded by NIH Office of Research Infrastructure Programs P40 OD010440, University of Minnesota) for providing strains.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

‘This work was funded by the Agence Nationale de la Recherche (grants ANR-10-LABX0030-INRT to Gilles Charvin and ANR-10-IDEX-0002; ANR 20-SFRI-0012; ANR-17-EURE0023 to the Interdisciplinary Thematic Institute IMCBio of the University of Strasbourg, CNRS and Inserm, part of the 2021-2028 Investments for the Future Program.

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Decision Letter 0

Elise A Kikis

29 Apr 2022

PONE-D-22-08767Distinct mechanisms underlie H2O2 sensing in C. elegans head and tailPLOS ONE

Dear Dr. QUINTIN,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. I encourage you to consider the comments of each review, with particular focus on some of the questions of reviewer 1. Specifically, certain aspects require more explanation, including how the identity of neurons was determined. This reviewer also raised some concerns about the statistical analyses and questions whether t-test are always the right test. Please also consider the suggestion by reviewer 2 to genetically dissect, using mutants or RNAi, the mechanism by which pmk-1 is required for H2O2 sensing.

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: The work of Quintin et al. examines the role of hydrogen peroxide sensing at the head and tail of C. elegans. The I2 pharyngeal neurons in the head and phasmid PHA neurons in the tail can both respond to H2O2, but with different sensitivities. The PHA neurons are more sensitive to micromolar concentrations whereas the I2 neurons require milimolar levels. The peroxiredoxin PRDX-2 is required in both cases, but may regulate detoxification in the I2 neurons and ROS signaling in the PHA neurons. The receptors GUR-3 and LITE-1 were found to play a role in light sensing and hydrogen peroxide sensing in the I2 neurons and PHA neurons, respectively. The paper provides insight into how animals can respond to the same signal through different mechanisms. Overall the experiments add to the field but there are some areas for improvement.

The authors measure changes in prdx-2::GFP expression in response to H2O2 in different cell types. How did the authors confirm the localization/identity of the neurons?

The concentrations of H2O2 differ experiments and range from 10mM, 1mM and 10uM. Please provide rationale for changing the concentrations in each experiment. Also, a discussion on the biological relevance of these concentrations would be helpful for the reader.

Statistical analysis should be improved. For some figures, a t-test is not appropriate. Welsh’s t-test should be corrected to “Welch”

Figure 1 – The authors show an increase in signal following exposure to H2O2 in the intestinal cells, which the authors attribute to increased expression of prdx-2::GFP. However, it appears that the time hours of 1-2 hours may not be sufficient to induce protein translation and the signaling could be an artifact of gut granule autofluorescence in response to the H2O2. The authors should demonstrate the selectivity of the single for prdx-2. In addition, the supplemental data in WT control should be quantified (2A and B).

From Figure 1, the authors make conclusions on the role of prdx-2 in I2 neurons based on expression changes (or lack thereof). While I agree that the responses of prdx-2 to oxidative stress are most likely “cellular context dependent”, the author’s conclusion is solely based on a GFP signal, which does not report on prdx-2 activity. Thus the authors should not overstate their conclusion.

The article has grammatical issues and unfinished sentences. As a note, the supplemental tables are not represented as tables. Genomic nomenclature should be followed throughout – sometimes the gene is listed when referring to the protein and vice versa. Also some of the titles do not fit with the data. One example, “Expression pattern of prdx-2 and its evolution upon H2O2 treatment.” How is prdx-2 evolving with H2O2 exposure? I would recommend editing throughout.

Figure 2 – The wormbase analysis seems more suited for the supplement since this is not new data. Moreover, it is unclear if the authors are permitted to reuse the data in their figure.

Figure 5 – The hypothetical model provided in C and D are helpful and supported by the data provided. However, A and B do not add any value and are confusing since the authors did not test or discuss any specific cysteine residues in their protein target. Additionally, it appears that the cysteine residues do not match with the protein sequences provided on wormbase.

The discussion is very detailed and often meanders from discussing the findings of the manuscript. I would suggest condensing the discussion to avoid over interpretation.

Reviewer #2: The manuscript submitted by Quintin et al identified how oxidants such as hydrogen peroxide (H2O2)are perceived by the I2 pharyngeal neurons and the tail phasmid PHA neurons in C. elegans. Using a GFP knock-in line for the peroxiredoxin PRDX-2, the authors demonstrate this peroxiredoxin functions as a putative H2O2 senor in both I2 and PHA neurons, while in the anterior gut and excretory pore cell it functions mainly as a peroxidase. Furthermore, the activation of the I2 and PHA neurons were dependent on the concentration of H2O2 administered which led to the identification of distinct molecular mechanisms to explain differences in sensitivity. Gustatory G-protein-coupled receptors GUR-3 and LITE-1 were shown to be expressed and required for the perception of H2O2 in the I2 and PHA neurons respectively. Further investigation using microfluidics and calcium imaging revealed PHA neurons required the p38 MAPK pathway in sensing H2O2 which has been shown to activate the voltage gated calcium channel OSM-9 in ASH neurons in the worm. Finally, in addition to sensing H2O2 the PHA neurons exhibited a slow photo-response to light, while the I2 neurons exhibited a fast response. The photo-response is dependent on PRDX-2 in the I2 neurons. However, in the PHA neurons PRDX-2 is not required. The manuscript is well written and addresses an important question in the field.

Concerns

1. It is thought that dimerized peroxiredoxin can interact with ASK-1 (NSY-1) to activate the p38 MAPK. Using calcium imaging the authors show pmk-1 is required for the sensing of H2O2 and suggest the p38 MAPK pathway is involved in this mechanism. However, the sek-1 and the nsy-1 mutant strains weren't included in the study. Including calcium measurements in the sek-1 and nsy-1 mutant strains would further support this claim.

2. According to the Li et al (2016) AKT-1 acts downstream of the p38 MAPK to phosphorylate OSM-9 in ASH neurons in response to H2O2 stimulation. Does Akt-1 act downstream of PMK-1 to activate OSM-9 in the PHA neurons? This could be tested using calcium imaging or speculated in the model. Furthermore, SKN-1 is also known to act downstream of the p38 MAPK. Could SKN-1 be a potential downstream activator if AKT-1 is not required in H2O2 sensing?

********** 

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Reviewer #2: No

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PLoS One. 2022 Sep 29;17(9):e0274226. doi: 10.1371/journal.pone.0274226.r002

Author response to Decision Letter 0


10 Aug 2022

Rebuttal Letter on Plos One Manuscript #PONE-D-22-08767

Summary of revisions

We thank the reviewers for their very careful evaluation of our manuscript. We took into consideration their comments, which we found all relevant. Specifically, the reviewers suggested to perform complementary analyses and controls, including more appropriate statistical analyses than a systematic use of t-test. To address Reviewer 2’s major concern, we added new data: we analyzed another mutant of the p38MAPK pathway, the sek-1 MAPKKK mutant. Performing these analyses further strengthened our conclusion that the p38MAPK pathway is required in PHA neurons for sensitivity to low doses of H2O2 (see new Fig 4, panels D-F). Furthermore, we systematically re-did all statistical analyses taking into account the multiple comparisons problem. Importantly, performing these more stringent statistical analyses did not change our conclusions.

For these reasons, we think that our revised manuscript has significantly improved compared to its initial version, and we believe that it fits PLOS ONE’s publication criteria.

Figures have been revised as follows:

— Figure 1 : panels E-F have been updated and include more data points, to reinforce the point that an increased PRDX-2::GFP signal is only observed in the gut upon a 10mM H2O2 treatment.

— Figure 2: panels C-D have been updated (multiple comparison statistical tests) and panel E has been transferred to a supplementary figure (S3 Fig), as it contained data retrieved from WormBase (addressing Reviewer 1’s point 7).

— Figure 3: panels H and L have been updated with the multiple comparison statistical tests, and their legends have been modified accordingly (addressing R1 point 3); panel K has been updated with 2 additional movies.

— Figure 4: panels D-F include the new data obtained with sek-1 MAPKK mutants analyses (addressing R2 point 1) and revised statistical analyses (R1 point 3).

— Figure 5: the hypothetical models from panels A-B have been moved to a supplementary figure (S12 Fig) to address R1 point 8.

Supplementary Figures have been revised as follows:

— S1 Fig includes new panels (F-H) to ascertain the identity of PRDX-2-expressing PHA/PHB neurons (addressing R1 point 1)

— S2 Fig includes a new panel (C) corresponding to the quantification of the gut autofluorescence in control animals treated with H2O2 (addressing R1 point 4), demonstrating the specificity of the PRDX-2::GFP signal.

— S3 Fig has been added, corresponding to the former bottom panel of Figure 2, showing the presumptive transcription factors binding sites in the promoter of prdx-2 (addressing R1 point 7).

— S10 Fig has been added, showing the individual fluorescence intensity measurements of the new supplementary sek-1 movies, and illustrates the lack of response of sek-1 mutants to 10μM H2O2,as observed in pmk-1 mutants (addressing R2 point 1).

— S13 Fig has been added, corresponding to the former top panel of Figure 5 (addressing R1 point 8). It also includes a new panel (A) with an alignment between GUR-3 and LITE-1 proteins, showing their conserved intracellular cysteines, further reinforcing the presumptive receptor activation model proposed in the discussion.

Supplemental material has been added, to support the new data included in the revised version (especially Figure 4):

— movie 16 (related to S10 Fig) - sek-1 mutant I2 response to 1mM H2O2

— movie 17 (related to S10 Fig) - sek-1 mutant PHA response to 1mM H2O2

— movie 18 (related to S10 Fig) - sek-1 mutant I2 response to 10μM H2O2

— movie 19 (related to S10 Fig) - sek-1 mutant PHA response to 10μM H2O2

S1-S3 Tables have been reorganized to address R1 point 6.

Concerning the References list, note that we have included 2 new papers: Zhang et al., 2022 (ref 62), which came out while we were working on revisions. It has been cited in the Discussion section (p. 11) for the reasons explained below (see R2 point 2 response). We have also added Quintin and Charvin (ref 73) in the Result section (p. 6), which has been submitted in the meantime and is currently under revision at micropublication Biology. This study, which describes a left-right asymmetry in I2 and PHA neurons, justified our approach to analyze individually left and right I2 and PHA neurons.

Please find below the original reviews as well as our reply/ comments in bold.

Reviewer #1: The work of Quintin et al. examines the role of hydrogen peroxide sensing at the head and tail of C. elegans. The I2 pharyngeal neurons in the head and phasmid PHA neurons in the tail can both respond to H2O2, but with different sensitivities. The PHA neurons are more sensitive to micromolar concentrations whereas the I2 neurons require milimolar levels. The peroxiredoxin PRDX-2 is required in both cases, but may regulate detoxification in the I2 neurons and ROS signaling in the PHA neurons. The receptors GUR-3 and LITE-1 were found to play a role in light sensing and hydrogen peroxide sensing in the I2 neurons and PHA neurons, respectively. The paper provides insight into how animals can respond to the same signal through different mechanisms. Overall the experiments add to the field but there are some areas for improvement.

We thank R1 for his globally positive evaluation of our work, and for his/her suggestions for improvement. However, we were a bit puzzled to read that R1 wrote that PRDX-2 ‘may regulate detoxification in the I2 neurons and ROS signaling in the PHA neurons’. We understand from this comment that our message was not fully clear, as we clearly think that PRDX-2 is involved in ROS signaling also in I2 neurons. We have tried to make this more clear in this revised version (see Results, page 5 and Discussion, page 9).

Point 1- The authors measure changes in prdx-2::GFP expression in response to H2O2 in different cell types. How did the authors confirm the localization/identity of the neurons?

We agree with R1 that neuron identification can sometimes be difficult in C. elegans. However, here, it was not the case for the reasons detailed below.

Regarding I2 neurons: as stated in the manuscript (page 4), previous work already reported the expression pattern of a PRDX-2 reporter transgene, especially in I2 neurons (Isermann et al., 2004 ; Bhatla and Horvitz, 2015). Importantly, a functional validation of this finding has been performed by mutant rescue analyses (Bhatla and Horvitz, 2015). The authors demonstrated that the light-induced feeding inhibition defect of gur-3 and prdx-2 mutants are both rescued by specifically restoring gur-3 and prdx-2 functions in I2 neurons, using an I2-specific promoter (termed “I2::gur-3” and “I2::prdx-2”), derived from the flp-15 gene (Kim and Li, 2004). Therefore, as GUR-3 and PRDX-2 functions are required in I2 neurons, there is no doubt on the identity of these head neurons.

Regarding CAN and PHA/PHB neurons; we indeed report for the first time expression of PRDX-2 in CAN and PHA/PHB tail neurons. The first assumptions were made by comparing the localization of PRDX-2::GFP positive neurons with neurons maps available at Wormatlas.org, as explained below.

— CAN neurons were easy to identify, as they are the only neurons positioned on each side midway along the body, labeled by PRDX-2::GFP (see Fig 1). Two lines of evidence support this observation (shown below): prdx-2 transcripts were abundantly detected in CAN neurons (Cao et al. 2017), and CAN neurons exhibit a very strong oxidized/reduced HyPer signal following exposure to H2O2 (Back et al, 2012), suggesting that these neurons are very sensitive to H2O2, as expected for PRDX-2 expressing cells.

This is indicated in the manuscript (p. 4): “Consistent with this, a high number of prdx-2 transcripts was detected in CANs and in the EPC (Cao et al, 2017).

— Regarding PHA/PHB neurons: their identification was made easier due to the fact that there are much fewer neurons in the tail than in the head. Based our initial observations, PRDX-2::GFP candidate neurons could have been DVA, DVB or DVC from the dorso-rectal ganglion, or anterior-most neurons from the lumbar ganglion, ie PHA, PHB (named phasmids) or PVQ, as illustrated below (Wormatlas images). Due to the rather posterior and ventral position of PRDX-2::GFP neurons right above the rectum slit, dorso-rectal ganglion neurons were excluded.

To discriminate between the remaining candidates (phasmids vs PVQ interneurons), a DiI filling assay was done in the PRDX-2::GFP knock-in line (a common method in C. elegans to stain amphid and phasmid sensory neurons, which are exposed to the environment). As PRDX-2::GFP neurons were stained by the red lipophilic dye, this validated the neuronal PHA/PHB identity (see below, we added this data to S1 Fig).

As a last piece of evidence, the GcAMP reporter we used, driven by the flp-15 promoter (specific to I2 and PHA neurons, Kim and Li, 2004) marks the same cells as PRDX-2::GFP (shown below).

In conclusion, we are certain about the identity of PRDX-2::GFP-expressing neurons, however we preferred not to include such a level of detail in the supplementary data as they already comprise 13 figures and 24 movies. As referenced on page 4, we included a new panel in S1 Fig (F-H), showing the DiI staining.

Point 2- The concentrations of H2O2 differ experiments and range from 10mM, 1mM and 10uM. Please provide rationale for changing the concentrations in each experiment. Also, a discussion on the biological relevance of these concentrations would be helpful for the reader.

We agree with R1 that this point deserved a clarification.

The choice of H2O2 concentrations was based on previous work, describing the animal’s response to various doses of H2O2. A sentence has been added in the manuscript (p.4) : ‘We selected H2O2 concentrations inducing different physiological responses (Bhatla and Horvitz, 2015). Interestingly, our observations regarding PRDX-2::GFP higher expression in the gut upon a 10mM treatment can find an explanation in the different behavior reported, as discussed later in the same paragraph (see p. 5, top).

Regarding neuron response analyses, we chose 1mM and 10μM again on the basis of the difference reported in animals’ physiological response: whereas at 1mM H2O2 nearly all animals stop pharyngeal pumping, the 10μM dose induces a less penetrant response, which we precisely aimed to test. We modified the sentence on p. 6 as follows:

While it was initially stated: ‘We thus tested whether I2 and PHA neurons exhibit a response to the very mild dose of 10μM H2O2. Indeed, at this dose, it has been reported that only a minority of animals (� 35%) respond by inhibiting pharyngeal pumping (Bhatla and Horvitz, 2015)’ ; the sentence was changed as follows: ‘We thus tested whether I2 and PHA neurons exhibit a response to the very mild dose of 10μM H2O2, a dose which induces a less penetrant pharyngeal pumping inhibition (� 35% of animals) than that observed at 1mM (� 90% of animals, Bhatla and Horvitz, 2015).

Point 3- Statistical analysis should be improved. For some figures, a t-test is not appropriate.

We thank again R1 for the major improvement suggested here.

For pairwise comparisons of data sets with a normal distribution (or N>30), p-values were calculated using an unpaired two-tailed Student test, and the Welch correction was applied when samples variance was not homogeneous. When distributions were not normal (Anderson-Darling and Shapiro Wilk tests not satisfied), a Mann Whitney test was used. Multiple comparisons were analyzed using a one-way ANOVA with Bonferroni’s correction (for normal distributions), or a non-parametrical Kruskal-Wallis test followed by a Dunn’s multiple comparisons test. Importantly, performing these new statistical analyses on our data did not change the validity of our conclusions. These modifications have been indicated in the paragraph describing statistical analyses (page 15) and in Figure legends, accordingly.

Welsh’s t-test should be corrected to “Welch”

Done.

Point 4- Figure 1 – The authors show an increase in signal following exposure to H2O2 in the intestinal cells, which the authors attribute to increased expression of prdx-2::GFP. However, it appears that the time hours of 1-2 hours may not be sufficient to induce protein translation and the signaling could be an artifact of gut granule autofluorescence in response to the H2O2. The authors should demonstrate the selectivity of the single for prdx-2. In addition, the supplemental data in WT control should be quantified (2A and B).

Regarding the possibility that PRDX-2::GFP could respond later than the examined time point, in our initial experiments we did not observe any significant difference in PRDX-2::GFP level when animals were analyzed 3-4 hours vs 1-2 hours after H2O2 treatment. Notably, in yeast, the peroxiredoxin reporter Tsa1-GFP (equivalent of PRDX-2) shows rapid induction with a peak within the 100 first minutes following the H2O2 stress (Goulev et al., 2017). To demonstrate the specificity of the PRDX-2 GFP signal, we are providing a quantification of H2O2-treated controls, which do not exhibit higher gut granule fluorescence after 10mM H2O2 treatment, in contrast to PRDX-2::GFP animals. This control quantification panel has been added in the revised version (S2 Fig, panel C). We are grateful to R1 for suggesting this improvement, which strengthens our conclusions.

Point 5- From Figure 1, the authors make conclusions on the role of prdx-2 in I2 neurons based on expression changes (or lack thereof). While I agree that the responses of prdx-2 to oxidative stress are most likely “cellular context dependent”, the author’s conclusion is solely based on a GFP signal, which does not report on prdx-2 activity. Thus the authors should not overstate their conclusion.

We agree with R1 that this conclusion should not be overstated, relative to the actual experiments. We indicated this limitation in our paragraph conclusion in the following manner (p. 5):

‘Although the induction we observed is only based on expression level and not on protein activity, we suggest that PRDX-2 could still scavenge H2O2 in the EPC and in the foregut, consistent with the reported protective role of intestinal PRDX-2 (Oláhová et al., 2008).’

Point 6- The article has grammatical issues and unfinished sentences. As a note, the supplemental tables are not represented as tables. Genomic nomenclature should be followed throughout – sometimes the gene is listed when referring to the protein and vice versa. Also some of the titles do not fit with the data. One example, “Expression pattern of prdx-2 and its evolution upon H2O2 treatment.” How is prdx-2 evolving with H2O2 exposure? I would recommend editing throughout.

We thank again R1 for helping us to improve the quality of the writing. We have changed the paragraph title into : ‘Expression pattern of PRDX-2::GFP and its variation upon H2O2 treatment’

Genomic nomenclature has been addressed as it should be in the C. elegans field, ie with gene names in small letters and proteins in capital letters (this does not apply to yeast Yap1). We modified the supplemental material so that only true tables are named tables (ie Tables S1-S3), while strain and primers lists are no longer called Tables but just ‘lists’. In addition, the revised manuscript has been extensively proof-read.

Point 7- Figure 2 – The wormbase analysis seems more suited for the supplement since this is not new data. Moreover, it is unclear if the authors are permitted to reuse the data in their figure.

We agree with R1 that this figure panel was more appropriate in a supplementary figure, as it was not based on our own findings. As commonly practiced in the literature related to C. elegans, we incorporated a screenshot arising from our data mining of Wormbase, properly citing the people who generated the raw data (in this case, Niu et al, 2011) and acknowledging WormBase (p.16). The panel has been moved to S3 Fig.

Point 8- Figure 5 – The hypothetical model provided in C and D are helpful and supported by the data provided. However, A and B do not add any value and are confusing since the authors did not test or discuss any specific cysteine residues in their protein target. Additionally, it appears that the cysteine residues do not match with the protein sequences provided on wormbase.

We appreciated R1’s positive evaluation of our model (formerly Fig 5CD). Regarding the presumptive PRDX-2-mediated receptor activation (formerly Fig 5AB), we agree with R1 that the model is speculative, and has not been tested experimentally. Still, we think that it has conceptual value, as it was inspired by an important literature survey and discussions with experts in the yeast Redox community (especially Dr Jacquel, acknowledged p. 16).

The PRDX-2 activation model has therefore been moved to a new supplementary figure (S12 Fig). In addition, we included a protein alignment between GUR-3 and LITE-1 receptors (panel A), to illustrate that conserved cysteines are only present in intracellular or transmembrane domains, thus supporting a presumptive intracellular activation by PRDX-2. Cysteine numbering has been corrected —we are grateful to R1 for noticing such a detail.

Point 9- The discussion is very detailed and often meanders from discussing the findings of the manuscript. I would suggest condensing the discussion to avoid over interpretation.

We condensed the discussion as much as we could, and did our best to avoid over interpretation. We especially attempted to place our interpretations in the context of a meaningful review of the relevant literature, also benefiting from the input of our Redox specialist colleagues. Thus, we feel that many of our discussion points are relevant and fill some gaps in the field. Therefore, we hope to have reached a satisfactory level of concision, while still highlighting biological connections that are important to understand this biological system.

Reviewer #2: The manuscript submitted by Quintin et al identified how oxidants such as hydrogen peroxide (H2O2)are perceived by the I2 pharyngeal neurons and the tail phasmid PHA neurons in C. elegans. Using a GFP knock-in line for the peroxiredoxin PRDX-2, the authors demonstrate this peroxiredoxin functions as a putative H2O2 senor in both I2 and PHA neurons, while in the anterior gut and excretory pore cell it functions mainly as a peroxidase. Furthermore, the activation of the I2 and PHA neurons were dependent on the concentration of H2O2 administered which led to the identification of distinct molecular mechanisms to explain differences in sensitivity. Gustatory G-protein-coupled receptors GUR-3 and LITE-1 were shown to be expressed and required for the perception of H2O2 in the I2 and PHA neurons respectively. Further investigation using microfluidics and calcium imaging revealed PHA neurons required the p38 MAPK pathway in sensing H2O2 which has been shown to activate the voltage gated calcium channel OSM-9 in ASH neurons in the worm. Finally, in addition to sensing H2O2 the PHA neurons exhibited a slow photo-response to light, while the I2 neurons exhibited a fast response. The photo-response is dependent on PRDX-2 in the I2 neurons. However, in the PHA neurons PRDX-2 is not required. The manuscript is well written and addresses an important question in the field.

We sincerely thank R2 for his/her very positive review.

Concerns

1. It is thought that dimerized peroxiredoxin can interact with ASK-1 (NSY-1) to activate the p38 MAPK. Using calcium imaging the authors show pmk-1 is required for the sensing of H2O2 and suggest the p38 MAPK pathway is involved in this mechanism. However, the sek-1 and the nsy-1 mutant strains weren't included in the study. Including calcium measurements in the sek-1 and nsy-1 mutant strains would further support this claim.

We agree with R2 that our initial conclusion on the p38MAPK pathway requirement in PHA hypersensitivity to H2O2 relied solely on the analysis of a single mutant, the pmk-1/MAPK mutant. As suggested, we further assessed the involvement of this pathway in PHA neurons by introducing the GcAMP calcium sensor in a sek-1/MAPKK mutant background (SXB70 new strain) to monitor I2 and PHA responses to H2O2. We observed that I2 neurons in sek-1 mutants responded normally to 1mM (movie 16), but not to 10μM H2O2 (movie 18), as in controls and as in pmk-1 mutants. Similarly, sek-1 mutants PHA neurons responded normally to 1mM H2O2 (movie 17), while pmk-1 mutant showed a slight diminution compared to WT. Importantly, in sek-1 mutants, as in pmk-1 mutants, PHA neurons were no longer able to respond to the low dose of 10μM H2O2 (movie 19), unlike in controls. Thus, our new analyses reveal that sek-1 mutants almost completely phenocopied pmk-1 mutants in their response to H2O2, further supporting the point that the p38MAPK pathway seems indeed to be specifically involved in PHA hypersensitivity to H2O2.

These new results have been incorporated in the revised version of Figure 4 (panels D-F); and they are supported by the addition of supplementary material (movies 16-19 and S10 Fig, depicting their quantification). The text has been accordingly modified in the manuscript in the results sections (paragraph now entitled ‘PMK-1 and SEK-1 are required for PHA neurons response to micromolar H2O2 but are dispensable in I2 neurons’, p. 7-8) and in the discussion (p. 9). Given that the C. elegans p38-MAPK pathway is linear (Inoue et al., 2005), and that sek-1 mutants showed the same defects as PMK-1 in PHA neurons, we considered that testing additional components of the same pathway (such as the nsy-1 MAPKKK) was not necessary here.

2. According to the Li et al (2016) AKT-1 acts downstream of the p38 MAPK to phosphorylate OSM-9 in ASH neurons in response to H2O2 stimulation. Does Akt-1 act downstream of PMK-1 to activate OSM-9 in the PHA neurons? This could be tested using calcium imaging or speculated in the model. Furthermore, SKN-1 is also known to act downstream of the p38 MAPK. Could SKN-1 be a potential downstream activator if AKT-1 is not required in H2O2 sensing?

We thank R2 for raising this hypothesis, highlighting his/her interest in our work. Li et al. (2016) indeed provided evidence that the TRPV channel OSM-9 can be phosphorylated in vitro by the AKT-1 kinase (on OSM-T10), and that this T10 residue has a key function in vivo, in potentiating the micromolar H2O2-induced sensory response in ASH neurons. It is tempting to speculate that a similar mechanism could take place in PHA neurons to promote their sensitivity to micromolar H2O2, but we have not tested it. As suggested by R2, this has been speculated in the discussion (p. 10-11).

Interestingly, while we were working on the revisions, the same group published an article showing that DLK/p38MAPK signaling regulates LITE-1 photoreceptor’s stability in ASH photosensory neurons (Zhang et al., 2022). Therefore, the involvement of PMK-1 in LITE-1 recycling in PHA neurons, as PMK-3 in ASH neurons, is another possibility. This has also been mentioned in the discussion (p. 11). All these questions should be addressed in the future, including the SKN-1 possible role proposed by R2.

Attachment

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Decision Letter 1

Elise A Kikis

24 Aug 2022

Distinct mechanisms underlie H2O2 sensing in C. elegans head and tail

PONE-D-22-08767R1

Dear Dr. QUINTIN,

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The authors have adequately addressed the reviewers comments and concerns, resulting in a stronger and study and clearer manuscript.

Reviewers' comments:

Acceptance letter

Elise A Kikis

6 Sep 2022

PONE-D-22-08767R1

Distinct mechanisms underlie H2O2 sensing in C. elegans head and tail

Dear Dr. Quintin:

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Related to Fig 1- The available PRDX-2::GFP transgenic line (CTD1051.3) does not suit to our study.

    (A) Low magnification fluorescent image of untreated animals from the CTD1051.3 line showing that transgenics express aggregates of PRDX-2::GFP, a hallmark of overexpression of the fusion protein. (B,C) Low magnification images of animals treated in flat bottom wells imaged after 24h of treatment in the potent oxidative stress inducing agent tBOOH. In the wild-type control well (A), almost all animals are all dead in 2mM tBOOH, appearing as rods, while CTD1051.3 transgenics survive a 25X higher dose of the drug, indicating their much stronger resistance to oxidative stress. (D) Representative image of an animal of the PRDX-2::GFP knock-in line; a wild-type control imaged with the same settings is shown (E), delineated by a dotted line. Bar, 100μm. (F-H) Confocal projections showing the tail region (dotted contours) of a PRDX-2::GFP transgenic animal stained with the lipophilic orange-red dye DiI (see https://www.wormatlas.org/EMmethods/DiIDiO.htm), imaged in green (F) and red channels (G). The overlay (H) shows that PRDX-2::GFP tail neurons are stained by the dye, establishing their identity as phasmid sensory neurons (PHA/PHB). Bar, 10μm.

    (TIF)

    S2 Fig. Related to Figs 1 and 2- PRDX-2 induction is not observed in I2 neurons upon H2O2 treatment.

    (A,B) Wild-type controls do not show a higher gut autofluorescence after H2O2 treatment, as illustrated by the int1 anterior gut cell fluorescence quantification (C). (D,E) Quantification of the PRDX-2::GFP fluorescence level in I2 neurons and in the excretory pore cell (EPC) in controls after 1mM-H2O2 treatment. (F,G) Quantification of I2 neurons’ PRDX-2::GFP fluorescence level in skn-1(zj15) mutants and in skn-1(RNAi) animals upon a 10mM-H2O2 treatment. Means are shown and error bars represent SD; ns, not significant, p>0.05 (t test or Mann-Whitney test). Scale bar, 50μm.

    (TIF)

    S3 Fig. Related to Fig 2.

    Genomic organization of the prdx-2 locus and presumptive regulation by SKN-1 and additional transcription factors. (A) Genome Browser screenshot from WormBase (release WS283, JBrowse II), showing the gene organization on chromosome II, at the indicated coordinates (top), and the peaks detected by ChIP-seq using an anti-GFP antibody [42] in prdx-2 and pkc-3 promoters, in GFP-tagged transgenic lines of the indicated transcription factors (B). Note the co-regulation of prdx-2 and pkc-3, which are organized in an operon, illustrated by the green bar.

    (TIF)

    S4 Fig. Related to Figs 3 and 4- Design of the microfluidic chip used in H2O2 neuron response experiments.

    (A) Global view of the microfluidic chip used in all neuron response experiments, showing the 6 independent series of 10 worm traps. The original file (Autocad format) is available at https://github.com/gcharvin/viewworm. (B) Magnification of the worm trap area showing the 10 individual channels (corresponding to the red box in A). Scale bar, 1mm. (C) DIC image of an animal trapped (anterior to the left). Occasionally animals were trapped in the opposite orientation, but this did not affect the neuronal response. Scale bar, 100μm.

    (TIF)

    S5 Fig. Related to Fig 3- Individual intensity measurements of S1S7 Movies.

    The curves represent the mean GCaMP3 intensity raw value over time (1 frame = 2sec) quantified in I2 and PHA neurons in control and prdx-2 mutants corresponding movies (S1S7 Movies), upon a 1mM or a 10μM H2O2 exposure. T0 indicate the time point at which the H2O2 treatment has been applied during 100 frames. Red and blue colors represent left and right I2 and PHA neurons (ie I2L/R and PHAL/R). Note that the colors have been changed in normalized average curves shown in Fig 3 and in related S6 Fig.

    (TIF)

    S6 Fig. Related to Fig 3- prdx-2 mutants PHA neurons do not respond to 10μM H2O2.

    (A-D) Average curves showing the normalized calcium response to 10μM H2O2 measured over time (in seconds) using the GCaMP3 sensor in PHA left and right neurons (top and bottom curves) in prdx-2(gk169) mutants (B) and in wild-type controls (A). N, number of movies analyzed for each genotype. See S6S7 Movies and related S5 Fig.

    (TIF)

    S7 Fig. Related to Fig 4- gur-3 and lite-1 mutants show reciprocal phenotypes in H2O2 sensing in I2 and in PHA neurons.

    (A-D) Average curves showing the normalized calcium response to 1mM H2O2 measured over time (indicated in seconds) using the GCaMP3 sensor in I2 and PHA left and right neurons (top and bottom curves) in gur-3(ok2245) (A,B) and lite-1(ce314) mutants (C,D). N, number of movies analyzed for each genotype. See S8S11 Movies and related S8 Fig.

    (TIF)

    S8 Fig. Related to Fig 4- Individual intensity measurements of S8S11 Movies (gur-3 and lite-1 mutants).

    The curves represent the mean GCaMP3 intensity raw value over time (1 frame = 2sec) quantified in I2 and PHA neurons in gur-3 and lite-1 mutants upon a 1mM H2O2 exposure (starting at T0 and lasting 100 frames), in corresponding movies (S8S11 Movies). Red and blue colors show left and right neurons for I2 (left panel) and PHA (right panel) neurons (ie I2L/R and PHAL/R). Note the reciprocal phenotypes observed in both mutants. Colors have been changed in the related normalized average curves shown in S7 Fig.

    (TIF)

    S9 Fig. Related to Fig 4, continued- Individual intensity measurements of S12S15 Movies (pmk-1 mutants).

    The curves represent the mean GCaMP3 intensity raw value over time (1 frame = 2sec) quantified in I2 and PHA neurons in pmk-1 mutants upon a 1mM or a 10μM H2O2 stimulation (starting at T0 and lasting 100 frames), in corresponding movies (S12S15 Movies). Red and blue color indicate I2L/R neurons (left panel) and PHAL/R neurons (right panel).

    (TIF)

    S10 Fig. Related to Fig 4, continued- Individual intensity measurements of S16S19 Movies (sek-1 mutants).

    The curves represent the mean GCaMP3 intensity raw value over time (1 frame = 2sec) quantified in I2 and PHA neurons in sek-1 mutants upon a 1mM or a 10μM H2O2 stimulation (starting at T0 and lasting 100 frames), in corresponding movies (S16S19 Movies). Red and blue color indicate left and right I2 (left panel) and PHA (right panel) neurons.

    (TIF)

    S11 Fig. Related to Fig 4, continued—Unlike in I2 neurons, PRDX-2 is not essential for light sensing in PHA neurons.

    Average curves showing the normalized calcium response to blue light over time (in seconds) using the GCaMP3 sensor, measured in the soma of I2 (A) and PHA neurons (B) in wild-type controls and in prdx-2(gk169) mutants. N, number of movies analyzed for each genotype. Error bars represent SD. While I2 neurons fail to respond to light in prdx-2 mutants, PHA neurons do respond, albeit with a lower intensity peak than in controls. See related S20S24 Movies.

    (TIF)

    S12 Fig. Related to Discussion- A presumptive model of how GUR-3 and LITE-1 receptors may be activated intracellularly by PRDX-2.

    (A) Alignment of LITE-1 and GUR-3 protein sequences made with the SIM alignment tool (https://web.expasy.org/sim/), using the comparison matrix BLOSUM30. Transmembrane (TM) and intra/extracellular domains were predicted using the DeepTMHMM program (https://dtu.biolib.com/DeepTMHMM). The alignment reveals 39.9% identity over 434 residues overlap. Note the rather large intracellular domains (in blue), encompassing conserved cysteines (boxed in red) (B-C) A putative PRDX-2 redox relay may trigger H2O2-induced receptor activation in I2 and PHA neurons. Sketch of GUR-3 (B) and LITE-1 (C) receptors, deduced from A, depicting their conserved cysteines. Upon H2O2 exposure, oxidized PRDX-2 or its disulfide form (PRDX-2ox/S-S) could oxidize these cysteines, possibly forming a disulfide conjugate and/or inducing a conformation change, which would subsequently trigger receptor activation, and I2 or PHA neuron response.

    (TIF)

    S13 Fig. Related to Discussion- PHA and ASH neurons belong to the same neuronal cluster, and share partially overlapping transcriptomic signatures.

    (A) Uniform Manifold Approximation and Projection (UMAP) projection of parent cluster 28, from [65] in which ASH and PHA/PHB nociceptive neurons were found in distinct sub-clusters (Louvain clustering at 8 PCs and at 1.2 resolution). (B, C) Dot plot indicating for a selection of genes both the intensity of gene expression and the fraction of expressing cells in each sub-cluster (B), based on single-cell RNA-sequencing data from [38]. See Supplementary Information and related S1S3 Tables for exhaustive lists of genes expressed in each cluster.

    (TIF)

    S14 Fig

    (TIF)

    S1 Movie. (Related to Fig 3)- Wild-type I2 response to 1mM H2O2.

    On-chip 4D movie showing control I2 neurons response to 1mM H2O2 visualized using the GCaMP3 calcium sensor. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S5 Fig.

    (MP4)

    S2 Movie. (Related to Fig 3)- Wild-type PHA response to 1mM H2O2.

    On-chip 4D movie showing control PHA neurons response to 1mM H2O2 visualized using the GCaMP3 calcium sensor. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S5 Fig.

    (MP4)

    S3 Movie. (Related to Fig 3)- prdx-2 mutant I2 response to 1mM H2O2.

    On-chip 4D movie showing the absence of I2 neurons response to 1mM H2O2 in a prdx-2 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S5 Fig.

    (MP4)

    S4 Movie. (Related to Fig 3)- prdx-2 mutant PHA response to 1mM H2O2.

    On-chip 4D movie showing the absence of PHA neurons response to 1mM H2O2 in a prdx-2 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S5 Fig.

    (MP4)

    S5 Movie. (Related to Fig 3)- Wild-type I2 response to 10μM H2O2.

    On-chip 4D movie showing the absence of I2 neurons response to 10μM H2O2 in a control animal. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S5 Fig.

    (MP4)

    S6 Movie. (Related to Fig 3)- Wild-type PHA response to 10μM H2O2.

    On-chip 4D movie showing control PHA neurons response to 10μM H2O2 visualized using the GCaMP3 calcium sensor. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S5 Fig.

    (MP4)

    S7 Movie. (Related to S6 Fig)- prdx-2 mutant PHA response to 10μM H2O2.

    On-chip 4D movie showing the absence of PHA neurons response to 10μM H2O2 in a prdx-2 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S5 Fig.

    (MP4)

    S8 Movie. (Related to Fig 4)- gur-3 mutant I2 response to 1mM H2O2.

    On-chip 4D movie showing the absence of I2 neurons response to 1mM H2O2 in a gur-3 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S8 Fig and related average response in S7 Fig.

    (MP4)

    S9 Movie. (Related to Fig 4)- gur-3 mutant PHA response to 1mM H2O2.

    On-chip 4D movie showing PHA neurons response to 1mM H2O2 in a gur-3 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S8 Fig and related average response in S7 Fig.

    (MP4)

    S10 Movie. (Related to Fig 4)- lite-1 mutant I2 response to 1mM H2O2.

    On-chip 4D movie showing I2 neurons response to 1mM H2O2 in a lite-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S8 Fig and related average response in S7 Fig.

    (MP4)

    S11 Movie. (Related to Fig 4)- lite-1 mutant PHA response to 1mM H2O2.

    On-chip 4D movie showing the absence of PHA neurons response to 1mM H2O2 in a lite-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S8 Fig and related average response in S7 Fig.

    (MP4)

    S12 Movie. (Related to Fig 4)- pmk-1 mutant I2 response to 1mM H2O2.

    On-chip 4D movie showing I2 neurons response to 1mM H2O2 in a pmk-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S9 Fig.

    (MP4)

    S13 Movie. (Related to Fig 4)- pmk-1 mutant PHA response to 1mM H2O2.

    On-chip 4D movie showing PHA neurons response to 1mM H2O2 in a pmk-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S9 Fig.

    (MP4)

    S14 Movie. (Related to Fig 4)- pmk-1 mutant I2 response to 10μM H2O2.

    On-chip 4D movie showing the absence of I2 neurons response to 10μM H2O2 in a pmk-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S9 Fig.

    (MP4)

    S15 Movie. (Related to Fig 4)- pmk-1 mutant PHA response to 10μM H2O2.

    On-chip 4D movie showing the absence of PHA neurons response to 10μM H2O2 in a pmk-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S9 Fig.

    (MP4)

    S16 Movie. (Related to Fig 4)- sek-1 mutant I2 response to 1mM H2O2.

    On-chip 4D movie showing I2 neurons response to 1mM H2O2 in a sek-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S10 Fig.

    (MP4)

    S17 Movie. (Related to Fig 4)- sek-1 mutant PHA response to 1mM H2O2.

    On-chip 4D movie showing PHA neurons response to 1mM H2O2 in a sek-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S10 Fig.

    (MP4)

    S18 Movie. (Related to Fig 4)- sek-1 mutant I2 response to 10μM H2O2.

    On-chip 4D movie showing the absence of I2 neurons response to 10μM H2O2 in a sek-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S10 Fig.

    (MP4)

    S19 Movie. (Related to Fig 4)- sek-1 mutant PHA response to 10μM H2O2.

    On-chip 4D movie showing the absence of PHA neurons response to 10μM H2O2 in a sek-1 mutant. The movie shown corresponds to a Z-projection of all time points (acquired every 2 sec), which has been processed for re-alignment (using the Matlab Readworm_PHA code). Accelerated 60 times. See fluorescence intensity measurements of this movie in S10 Fig.

    (MP4)

    S20 Movie. (Related to Fig 4)- Wild-type control I2 response to light.

    Single-Z movie (recorded at 10 fps) showing control I2 neurons response to blue light (485nm), visualized with the GCaMP3 calcium sensor. The original movie has been colored artificially to highlight fluorescence intensity variations, using Fiji ‘glow’ look up table. Note the quasi instantaneous and very strong posterior neurite response. Accelerated 5 times.

    (AVI)

    S21 Movie. (Related to Fig 4)- Wild-type control PHA response to light.

    Single-Z movie (recorded at 10 fps) showing control PHA neurons response to blue light (485nm), visualized with the GCaMP3 calcium sensor. The original movies has been colored artificially to highlight fluorescence intensity variations, using Fiji ‘glow’ look up table. Note the long-lasting PHA soma response. Accelerated 5 times.

    (AVI)

    S22 Movie. (Related to Fig 4)- Wild-type control I2 and PHA simultaneous responses to light.

    Single-Z movie (recorded at 10 fps) showing simultaneously control I2 (right) and PHA (left) neurons response to blue light (485nm). The original movies has been colored artificially to highlight fluorescence intensity variations, using Fiji ‘glow’ look up table. Note the contrast of response, with the quasi instantaneous response of I2 neurons versus the slower and longer soma response in PHA neurons. Accelerated 5 times.

    (AVI)

    S23 Movie. (Related to Fig 4)- prdx-2 mutant I2 response to light.

    Single-Z movie (recorded at 10 fps) showing the absence of I2 neurons response to blue light (485nm) in a prdx-2 mutant, visualized with the GCaMP3 calcium sensor. The original movie has been colored artificially to highlight fluorescence intensity variations, using Fiji ‘glow’ look up table. Accelerated 5 times. See related average response in S11 Fig.

    (AVI)

    S24 Movie. (Related to Fig 4)- prdx-2 mutant PHA response to light.

    Single-Z movie (recorded at 10 fps) showing PHA neurons response to blue light (485nm) in a prdx-2 mutant, visualized with the GCaMP3 calcium sensor. The original movie has been colored artificially to highlight fluorescence intensity variations, using Fiji ‘glow’ look up table. Note the long-lasting PHA soma response in prdx-2 mutant as in WT (S21 Movie). Accelerated 5 times. See related average response in S11 Fig.

    (AVI)

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    Submitted filename: Response_to_Reviewers.pdf

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.


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