Avoidance of hydrogen sulfide is modulated by external and internal states in Caenorhabditis elegans
- Department of Molecular Biology, Umeå University, Sweden
- Umeå Centre for Molecular Medicine, Umeå University, Sweden
- Wallenberg Centre for Molecular Medicine, Umeå University, Sweden
- Laboratory of Neurophysiology, ULB Neuroscience Institute (UNI), Université Libre de Bruxelles (ULB), Belgium
- The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Sweden
- Integrated Science Lab (Icelab), Umeå University, Sweden
Figures
Figure 1 with 1 supplement
Acute locomotory responses of C. elegans to hydrogen sulfide.
(A) Changes in the locomotory speed of WT animals (N2 laboratory strain) evoked by a switch from 7% O2 to 150 ppm H2S balanced with 7% O2, followed by a return to 7% O2. (B) Proportion of WT animals undergoing reorientation movements (omega turns) during a switch from 7% O2 to 150 ppm H2S balanced with 7% O2, followed by a return to 7% O2. (C) Fraction of WT animals doing backward locomotion (reversals) during a switch from 7% O2 to 150 ppm H2S balanced with 7% O2, followed by a return to 7% O2. (D) Locomotory speed of WT was recorded for 2 min at 7% O2, followed by 148 min at 150 ppm H2S balanced with 7% O2, and then at 7% O2. (E) Locomotory speed of WT animals in response to near-UV light (435 nm, 0.7 mW/mm2) exposure, before and directly after 30-min preincubation in 150 ppm H2S balanced with 7% O2. Locomotory activity was recorded for 2 min before, during, and after UV light exposure. (F) Locomotory speed of WT animals to different concentrations of H2S balanced with 7% O2. Red bars on the x-axis represent two intervals (4–5 min and 11–12 min, labeled a and b, respectively) used for statistical analysis. **** = p < 0.0001, ** = p < 0.01, * = p < 0.05, ns = not significant, Mann–Whitney U test. (G) Distribution of WT animals in a microfluidic device after 25 min of aerotaxis. The gas inputs and the five chamber sections used for scoring are indicated. (H) Distribution of WT animals in a gradient of H2S (150 ppm to 0 ppm). The bins correspond to different sections of the microfluidic chamber described in (G). N = 4 aerotaxis assays.
Figure 1—video 1
The locomotory response of WT animals to H2S.
An 8 x accelerated video clip (original length: 16 min) showing WT (N2) animals exposed to 7% O2 for 2 min, followed by 11 min in 150 ppm H2S, and then returned to 7% O2. The H2S exposure segment is highlighted and begins at the 15 s mark, corresponding to 2 min in the original recording.
Figure 2 with 2 supplements
Neurosecretion from ASJ neurons contributes to H2S avoidance.
(A–H) Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT and daf-19(m86)(A); WT and dyf-3(m185)(B); WT and dyf-7(m539)(C); WT, daf-11(m47), and tax-4(p678)(D); WT and tax-2(p691) (E); WT, daf-11(m47), and transgenic daf-11(m47) expressing daf-11 genomic DNA in ASJ neurons (two independent lines) (F); WT, tax-4(p678), and transgenic tax-4(p678) expressing tax-4 cDNA in ASJ neurons (two independent lines) (G); WT and transgenic WT expressing the catalytic domain of the tetanus toxin (TeTx) in ASJ neurons (H). For the comparison of acute locomotory speed responses between strains, red bars on the x-axis represent two intervals (4–5 min and 11–12 min, labeled a and b, respectively, in panels D, F, and G) used for statistical analysis. **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns = not significant, Mann–Whitney U test. (I) Calcium transients evoked in ASJ neurons of WT animals in response to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals (N=42). The slow decline in GCaMP6s fluorescence is due to photobleaching.
Figure 2—figure supplement 1
Acute avoidance response to H2S is regulated by cGMP signaling.
(A–H) Fraction of animals undergoing reorientation movements (omega turns, left) or backward locomotion (reversals, right) during a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT and daf-19(m86) (A, B); WT and dyf-3(m185) (C, D); WT and dyf-7(m539) (E, F); WT, daf-11(m47), and tax-4(p678) (G, H).
Figure 2—figure supplement 2
Acute response to H2S is regulated by daf-11 signaling in ASJ neurons.
(A) The detailed expression pattern of each promoter used to express daf-11 genomic DNA. Promoters shown in green rescued the daf-11(m47) mutant phenotype. (B–D) Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT, daf-11(m47), and transgenic daf-11 mutants expressing daf-11 genomic DNA in different subsets of neurons. Red bars on the x-axis represent two intervals (4–5 min and 11–12 min, labeled a and b, respectively) used for statistical analysis. * = p < 0.05, ns = not significant, Mann–Whitney U test. (E) Calcium transients evoked in ASJ neurons of WT animals in response to a switch from 7% O2 to 3% CO2 balanced with 7% O2 for animals (N=16).
Figure 3 with 1 supplement
Insulin, TGF-β, and high O2 signaling pathways antagonize H2S avoidance.
(A, B) Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT, daf-2(e1370), daf-16(mgDf47), and daf-2(e1370); daf-16(mgDf47) double mutants (A); WT, daf-7(e1372), daf-3(mgDf90), and daf-7(e1372); daf-3(mgDf90) double mutants (B). For the daf-2 assays, WT and daf-2 mutants were maintained at 15 °C. L4 animals were picked and shifted to 25 °C until day-1 adults, then assayed at room temperature. (C) Hypothetical model for the regulation of H2S avoidance by a starvation program involving the insulin, TGF-β, and NHR-49 pathways. (D) Locomotory speed response to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 in fed and starved WT animals. (E, F) Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT and wild isolates (CB4858 and CB4855) (E); WT and npr-1(ad609) (F). (G) Hypothetical model showing how O2 regulates H2S avoidance via the inhibitory RMG circuit activity, which is modulated by O2 sensory inputs and NPR-1 signaling. (H–L) Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT, npr-1(ad609), and transgenic npr-1(ad609) expressing npr-1 in RMG neurons using Cre-LoxP system (Macosko et al., 2009) (H); WT, npr-1(ad609), gcy-35(ok769), and npr-1(ad609); gcy-35(ok769) double mutants (I); WT, npr-1(ad609), tax-4(p678) and npr-1(ad609); tax-4(p678) double mutants (J); WT, npr-1(ad609), qaIs2241(genetic ablation of AQR, PQR, and URX neurons) and npr-1(ad609); qaIs 2241 (K); WT, npr-1(ad609), npr-1(ad609); gcy-35(ok769) double mutants and transgenic npr-1(ad609); gcy-35(ok769) double mutants expressing gcy-35 cDNA under gcy-37 promoter, which drives gcy-35 expression in O2 sensing neurons (L). For the comparison of acute locomotory responses between strains, red bars on the x-axis represent two intervals (4–5 min and 11–12 min, labeled a and b, respectively, in panel E) used for statistical analysis. ****=p < 0.0001, ***=p < 0.001, **=p < 0.01, *=p < 0.05, ns = not significant, Mann–Whitney U test.
Figure 3—figure supplement 1
Insulin, TGF-β, and O2 signaling modulate locomotory response to H2S.
(A–C) Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT and nhr-49(nr2041) (A); WT and tax-6(ok2065) (B); WT and cnb-1(ok276) (C). (D–F) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for WT and daf-2(e1370), including locomotory speed (D), reorientation (omega turns) (E), and reversal (F). WT and daf-2 mutants were maintained at 15 °C. L4 animals were picked and shifted to 25 °C until day-1 adults, then assayed at room temperature. (G–I) Locomotory responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for WT and gcy-9(tm7632), including locomotory speed (G), reorientation (omega turns) (H), and reversal (I). (J) Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for WT animals and wild isolate CB4856. (K) Optogenetic stimulation of RMG neurons using channelrhodopsin-2 (ChR2). ChR2 was expressed in RMG neurons using the Cre-LoxP system (Macosko et al., 2009). Blue light was delivered as soon as the assay was initiated at 7% O2. Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 were recorded for day-1 adults grown for 16 hr in the presence or absence of ATR. (L) Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2, following activation of O2 sensing neurons by expressing a gain-of-function allele of pkc-1 in O2 sensory neurons under gcy-37 promoter. For the comparison of acute locomotory speed responses between strains or conditions, red bars on the x-axis represent two intervals (4–5 min and 11–12 min, indicated with red bars on the x-axis) for statistical analysis. ****=p < 0.0001, ***=p < 0.001, **=p < 0.01, ns = not significant, Mann–Whitney U test.
Figure 4 with 1 supplement
Prolonged H2S exposure reprograms gene expression in C. elegans.
(A) Venn diagram displaying the number of differentially expressed genes after 1 hour exposure to 50 ppm or 150 ppm H2S balanced with 7% O2 in WT animals (adjusted p<1e-10). (B) Venn diagram displaying the number of differentially expressed genes after 1-, 2-, and 12 hr exposure to 150 ppm H2S balanced with 7% O2 in WT animals (adjusted p<1e-10). (C, D) Significantly enriched GO categories for differentially expressed genes with adjusted p<1e-10 in WT animals exposed to 50 ppm (C) or 150 ppm (D) H2S balanced with 7% O2 for 1 hr. (E, F) Volcano plots showing the differentially expressed genes with adjusted p<1e-10 in WT animals exposed to 50 ppm (E) or 150 ppm (F) H2S balanced with 7% O2 for 1 hr. A set of genes involved in H2S detoxification, cysteine metabolism, and stress response was highlighted in red.
Figure 4—figure supplement 1
Transcriptome reprogramming induced by H2S exposure.
(A) Venn diagram displaying the number of differentially expressed genes with adjusted p value <1e-10 in WT animals exposed to 50 ppm H2S balanced with 7% O2 for 1, 2, or 12 hr. (B, C) Significantly enriched GO categories for differentially expressed genes with adjusted p value <1e-10 in WT animals exposed to 50 ppm (B) or 150 ppm (C) H2S balanced with 7% O2 for 2 hr. (D, E) Volcano plots showing the differentially expressed genes with adjusted p value <1e-10 in WT animals exposed to 50 ppm (D) or 150 ppm (E) H2S balanced with 7% O2 for 2 hr. A set of genes involved in H2S detoxification, cysteine metabolism, and stress response is highlighted in red. (F, G) The number of HIF-1(F) and SKN-1(G) target genes induced by 50 ppm or 150 ppm H2S exposure for 1 hr, 2 hr, or 12 hr in WT animals, with adjusted p value <1e-30. (H–J) Venn diagrams displaying the number of differentially expressed genes (adjusted p value <1e-10) in WT animals under the indicated conditions: animals exposed to 50 ppm or 150 ppm H2S for 1 hr (H); 2 hr (I); and 12 hr (J). (K, L) Volcano plots showing the differentially expressed genes (adjusted p value <1e-10) in WT animals that were exposed to either 50 ppm (K) or 150 ppm (L) H2S balanced with 7% O2 for 12 hr. A set of genes involved in H2S detoxification, cysteine metabolism, and stress response is highlighted in red.
Figure 5 with 1 supplement
Acute response to H2S is modulated by HIF-1 signaling.
(A) Locomotory speed responses of WT animals to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 after incubation of the animals for 0, 12, 24, or 36 hr in 1% O2. (B, C) Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT, egl-9(sa307), and vhl-1(ok161) (B); WT and transgenic animals expressing the non-degradable form of HIF-1 (P621A or P621G) under the pan-neuronal unc-14 promoter or the endogenous hif-1 promoter, respectively (C). (D–F) Avoidance responses of WT and hif-1(ia4) to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2, including locomotory speed (D), reorientation (omega-turn) (E), and reversal (F). (G, H) Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT, egl-9(sa307), hif-1(ia4), and egl-9(sa307); hif-1(ia4) double mutants (G); WT, vhl-1(ok161), hif-1(ia4), and vhl-1(ok161); hif-1(ia4) double mutants (H). (I) Average locomotory speed during 2 min of exposure to near-UV light for WT, hif-1(ia4), and egl-9(sa307) before (Ctrl = control) and after 30 min of exposure to 150 ppm H2S balanced with 7% O2. (J–L) Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT and sqrd-1(tm3378) (J); WT and ethe-1(yum2895) (K); WT, cysl-1(ok762), cysl-2(ok3516), and cysl-3(yum4) (L). For the comparison of acute locomotory responses between strains, red bars on the x-axis represent two intervals (4–5 minutes and 11–12 minutes, labeled a and b, respectively, in panels A, B, C, and L) used for statistical analysis. ***=p < 0.001, **=p < 0.01, *=p < 0.05, ns = not significant, Mann–Whitney U test.
Figure 5—figure supplement 1
Acute response to H2S is modulated by HIF-1 signaling.
(A, B) Fraction of WT and egl-9(sa307) animals undergoing reorientation movements (omega turns) (A) or backward locomotion (reversals) (B) during a switch from 7% O2 to 150 ppm H2S balanced with 7% O2. (C, D) Locomotory speed responses to a switch from 7% O2 to 1% O2 for WT and egl-9(sa307) (C); WT, hif-1(ia4), and semo-1(yum2889) (D). (E) HIF-1 signaling regulates the H2S mitochondrial detoxification pathway. The detoxification enzyme SQRD-1 (sulfide:quinone oxidoreductase) oxidizes H2S, donating electrons to ubiquinone (uq), and rhodoquinone (rq) of the mitochondrial electron transport chain (ETC). ETHE-1 (persulfide dioxygenase) oxidizes sulfur compounds to sulfite. (F–I) Fraction of animals undergoing reorientation movements (omega turns, F and H) or backward locomotion (reversals, G and I) during a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for the indicated genotypes: WT and sqrd-1(tm3378) (F, G); and WT and ethe-1(yum2895) (H, I). (J) Locomotory speed responses to a switch from 7% O2 to 1% O2 for WT, sqrd-1(tm3378), and ethe-1(yum2895). (K, L) Fraction of WT, cysl-1(ok762), cysl-2(ok3516), and cysl-3(yum4) animals undergoing reorientation movements (omega turns, K) or backward locomotion (reversals, L) during a switch from 7% O2 to 150 ppm H2S balanced with 7% O2. (M) Locomotory speed responses to a switch from 7% O2 to 1% O2 for WT, cysl-1(ok762), cysl-2(ok3516), and cysl-3(yum4) animals. (N) Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for WT and semo-1(yum2889). (O, P) Fraction of animals undergoing reorientation (omega turns, O) or backward locomotion (reversals, P) in response to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2. Comparisons of acute locomotory responses between strains were performed using two intervals (4–5 min and 11–12 min, indicated with red bars on the x-axis) for statistical analysis. ***=p < 0.001, ns = not significant, Mann–Whitney U test.
Figure 6 with 1 supplement
Labile iron pool sustains the locomotory activity in H2S.
(A, B) Volcano plots showing the relative expression of the genes involved in the regulation of iron homeostasis in WT animals after 2 hr exposure in 50 ppm (A) or 150 ppm (B) H2S balanced with 7% O2. (C–G) Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of indicated genotypes or treatments: WT and animals overexpressing ftn-1 genomic DNA under its own promoter (#1 and #2 indicate two independent lines) (C); WT, smf-3(ok1305), and ftn-1(ok3625) (D); WT, and WT pretreated with 100 μM 2,2′-Bipyridyl (BP) or with 5 mg/ml ferric ammonium citrate (FAC) in the presence of food for 16 hr (E); WT, hif-1(ia4), and hif-1(ia4) mutants pretreated with 5 mg/ml ferric ammonium citrate (FAC) for 16 hr (F); WT, hif-1(ia4), ftn-1(ok3625), and hif-1(ia4); ftn-1(ok3625) double mutants (G). (H) Hypothetical model of the interactions between labile iron pool and HIF-1 signaling. For the comparison of acute locomotory speed responses between strains or conditions, red bars on the x-axis represent two intervals (4–5 min and 11–12 min, labeled a and b, respectively, in panels C, D, and E) used for statistical analysis. ****=p < 0.0001, ***=p < 0.001, **=p < 0.01, *=p < 0.05, ns = not significant, Mann–Whitney U test.
Figure 6—figure supplement 1
Labile iron pool sustains the locomotory activity in H2S.
(A–D) Volcano plots showing the relative expression of the genes involved in the regulation of iron homeostasis (ftn-1, ftn-2, and smf-3) in WT animals under the following conditions: 1 hr exposure in 50 ppm H2S balanced with 7% O2 (A); 1 hr exposure in 150 ppm H2S balanced with 7% O2 (B); 12 hr exposure in 50 ppm H2S balanced with 7% O2 (C); and 12 hr exposure in 150 ppm H2S balanced with 7% O2 (D). (E, F) Locomotory speed responses to a switch from 7% O2 to 1% O2 for animals of the indicated genotype or treatment: WT, and two independent transgenic lines overexpressing ftn-1 genomic DNA under its own promoter (#1 and #2) (E); WT animals, WT animals treated with 100 μM 2,2′-Bipyridyl (BP), and WT animals treated with 5 mg/ml ferric ammonium citrate (FAC) for 16 hours (F). Red bar on the x-axis represents 1-min time interval (3–4 minutes) used for statistical analysis. *=p < 0.05, ns = not significant, Mann–Whitney U test. (G, H) Fraction of animals undergoing reorientation movements (omega turns) (G) or backward locomotion (reversals) (H) during a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotypes WT, smf-3(ok1305), and ftn-1(ok3625). (I, J) Fraction of animals undergoing reorientation movements (omega turns) (I) or backward locomotion (reversals) (J) during a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for WT animals, WT animals pretreated with either 100 μM 2,2′-Bipyridyl (BP) or with 5 mg/ml ferric ammonium citrate (FAC) in the presence of food for 16 hr.
Figure 7 with 1 supplement
Mitochondrial function is required for acute response to H2S.
(A–D) Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT and gas-1(fc21) (A); WT and clk-1(qm30) (B); WT and mev-1(kn1) (C); WT and isp-1(qm150) (D). (E) Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for WT animals pretreated with 10 μM rotenone for 0 min, 10 min, 30 min, 60 min, or 120 min. For the comparison of acute locomotory speed responses between strains or conditions, red bars on the x-axis represent two intervals (4–5 min and 11–12 min) used for statistical analysis. ***=p < 0.001, **=p < 0.01, *=p < 0.05, ns = not significant, Mann–Whitney U test. (F–G) Locomotory speed responses to a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for animals of the indicated genotype: WT, sod-1(tm776), sod-2(ok1030), and sod-3(tm760) (F); WT, sod-4(gk101), sod-5(tm1146), and sod-1(tm783); sod-2(ok1030); sod-3(tm760); sod-4(gk101); sod-5 (tm1146) (G). For the comparison of acute locomotory speed responses between strains or conditions, red bars on the x-axis represent the time interval (6–10 min) used for statistical analysis. ***=p < 0.001, **=p < 0.01, *=p < 0.05, ns = not significant, Mann–Whitney U test. (H) Hypothetical model of the role of mitochondria in response to toxic levels of H2S. In this model, mitochondria play a dual role in H2S-evoked avoidance behavior. A transient burst of mitochondrial ROS triggered by high H2S levels initiates locomotory avoidance, whereas sustained ROS elevation activates stress-responsive pathways, including HIF-1, NRF2/SKN-1, and DAF-16/FOXO, promoting adaptation to prolonged H2S exposure.
Figure 7—figure supplement 1
Mitochondrial function is required for acute response to H2S.
(A–D) Fraction of animals undergoing reorientation movements (omega turns) or backward locomotion (reversals) during a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for the indicated genotype or treatment: WT and clk-1(qm30) (A, B); WT and mev-1(kn1) (C, D). (E) Locomotory speed responses to a switch from 7% O2 to 1% O2 for WT, sod-2(ok1030); and sod-3(tm760). (F, G) Fraction of animals undergoing reorientation movements omega turns, (F) or backward locomotion reversals, (G) during a switch from 7% O2 to 150 ppm H2S balanced with 7% O2 for WT and sod-1(tm783); sod-2(ok1030); sod-3(tm760); sod-4(gk101); sod-5 (tm1146). (H) Locomotory response to acute near UV exposure for WT and sod-1(tm783); sod-2(ok1030); sod-3(tm760); sod-4(gk101); sod-5 (tm1146) before and after 30 min of exposure to 150 ppm H2S.
Additional files
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Supplementary file 1
H2S-evoked behavioral responses in mutants from the candidate gene screen.
- https://cdn.elifesciences.org/articles/92964/elife-92964-supp1-v1.docx
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Supplementary file 2
RNA-seq analysis of gene expression responses to 50 or 150 ppm H2S at different time points.
- https://cdn.elifesciences.org/articles/92964/elife-92964-supp2-v1.xlsx
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Supplementary file 3
List of strains used in this study.
- https://cdn.elifesciences.org/articles/92964/elife-92964-supp3-v1.xlsx
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Supplementary file 4
List of reagents and primers used in this study.
- https://cdn.elifesciences.org/articles/92964/elife-92964-supp4-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/92964/elife-92964-mdarchecklist1-v1.pdf
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Avoidance of hydrogen sulfide is modulated by external and internal states in Caenorhabditis elegans
eLife 12:RP92964.
https://doi.org/10.7554/eLife.92964.4
