Avoidance of hydrogen sulfide is modulated by external and internal states in Caenorhabditis elegans

Animals’ behavior and physiology are profoundly influenced by the environments in which they evolved. The laboratory strain N2, which is adapted to low atmospheric CO2 and H2S concentrations, robustly avoids both gases (Beets et al., 2020; Bretscher et al., 2008; Carrillo et al., 2013; Hallem and Sternberg, 2008; Kodama-Namba et al., 2013; McGrath et al., 2009). Upon acute exposure to H2S above 75 ppm, N2 exhibits an avoidance behavior characterized by reorientation and increased locomotion. This response is significantly reduced in wild isolates. These divergent responses between the laboratory strain and wild isolates are primarily driven by variations in the npr-1 gene, which encodes a neuropeptide receptor, suggesting that C. elegans has undergone rapid evolutionary adaptation to its environment.

The behavioral response to a specific sensory stimulus is usually shaped by an interplay of multiple environmental and physiological cues. We observe that conditions dampening C. elegans’ response to CO2 also impair the response to H2S. Specifically, the speed response to H2S is significantly inhibited at high O2 levels, and in mutants with defective insulin or TGF-β pathway that signal starvation. However, despite these shared modulations, CO2 and H2S responses involve distinct molecular mechanisms. For example, CO2 responses are mediated by the guanylate cyclase GCY-9 in BAG neurons (Hallem et al., 2011), which is dispensable for H2S responses. In addition, acute exposure to H2S induces a delayed acceleration compared to CO2. These data suggest that while responses to the two gases share a downstream neural circuit that can be dynamically modulated by the state of O2 sensing circuit, they are triggered by distinct mechanisms.

In a candidate gene survey, we excluded the potential involvement of globins, K+ channels, biogenic amines, and most guanylate cyclases in H2S responses. However, we observed that H2S avoidance requires the activity of guanylate cyclase DAF-11 in ASJ neurons, as well as neurosecretion from these neurons. Since the DAF-11 pathway and neurosecretion from ASJ neurons regulate developmental programs that modify sensory functions in C. elegans (Murakami et al., 2001), it is not surprising that daf-11 mutants display pleiotropic phenotypes including impaired H2S and CO2 responses (Hallem and Sternberg, 2008). In addition, we did not detect an H2S-evoked calcium transient in ASJ neurons. Therefore, although ASJ neurons and DAF-11 activity are clearly required, how H2S avoidance is triggered remains to be elucidated.

Consistent with previous reports (Horsman et al., 2019; Miller et al., 2011), H2S exposure substantially alters the gene expression in C. elegans, including those involved in H2S detoxification and iron homeostasis pathways. The repression of ftn-1 and induction of smf-3 observed during H2S exposure suggests that H2S depletes intracellular labile iron. Iron is a critical cofactor required for the activity of many enzymes and supports a wide range of cellular processes (Dev and Babitt, 2017; Hentze et al., 2004). One important enzyme that requires labile iron as a cofactor is PHD/EGL-9, which targets HIF-1 for degradation. Changes in iron availability are therefore expected to modulate EGL-9 activity, which in turn alters the expression of HIF-1-regulated genes (Liochev, 1996; Myllyharju and Kivirikko, 1997; Read et al., 2021; Xu and Møller, 2011). However, ETHE-1, a key H2S detoxification enzyme upregulated upon HIF-1 stabilization, also requires iron for its persulfide dioxygenase activity (Kabil and Banerjee, 2012; Pettinati et al., 2015). Therefore, H2S-evoked responses under varying iron availability are determined by the combined effects of labile iron on HIF-1 signaling and on the H2S detoxification enzymes, among other factors. For instance, although reduced iron availability in smf-3 or BP-treated animals may promote the HIF-1 induced detoxification pathways by inactivating EGL-9, the actual enzymatic clearance of H2S is impaired due to low ETHE-1 activity. Supporting this, smf-3 mutants and BP-treated animals display increased sensitivity to H2S, including enhanced initial omega-turn responses and rapid inhibition of locomotion. In contrast, increased iron availability in ftn-1 mutants or FAC-treated animals likely delays iron depletion during H2S exposure, preserving ETHE-1 detoxification capacity and postponing the onset of HIF-1-mediated adaptations that are associated with reduced locomotion. This may explain the sustained high locomotory speed observed in ftn-1 mutants or FAC-treated animals. In the absence of HIF-1, iron supplementation only partially improves the locomotory response to H2S, likely because the H2S detoxification system including ETHE-1 cannot be transcriptionally induced. The modest effect observed may instead reflect correction of iron deficiency in hif-1 mutants.

At concentrations below 50 ppm, H2S is well tolerated or even preferred, with beneficial effects such as improved thermotolerance (Fawcett et al., 2015; Miller and Roth, 2007; Qabazard et al., 2014). Notably, behavioral avoidance is absent below 50 ppm H2S, suggesting that escape behavior is triggered only when H2S is not efficiently detoxified. This led us to hypothesize that animals with enhanced detoxification capacity would show reduced avoidance of otherwise toxic H2S levels. Indeed, the speed response to H2S is attenuated when the detoxification program is upregulated under conditions of constitutive activation of HIF-1, such as in egl-9 or vhl-1 mutants, after prolonged exposure to 1% O2, or when HIF-1 signaling is specifically activated in neurons. These observations support the idea that the initial avoidance response to H2S is triggered by neuronal detection of acute toxicity, while the subsequent decline in speed reflects a rapid activation of adaptive mechanisms, particularly through stabilization of HIF-1. The fact that wild-type animals remain responsive to other stimuli after prolonged H2S exposure suggests that reduced locomotory speed in H2S reflects active desensitization rather than general paralysis. However, persistently high H2S exposure is likely to exhaust cellular defense systems, leading to toxicity and paralysis. By contrast, the rapid loss of locomotion observed in hif-1 mutants and detoxification-defective mutants is mediated by a mechanism distinct from adaptation. Their loss of responsiveness to other stimuli after H2S exposure suggests that these animals likely become sensitized and rapidly intoxicated by H2S due to impaired detoxification. Therefore, H2S exposure promotes a behavioral program that includes an initial reorientation and acceleration responses followed by a progressive adaptation driven by cellular detoxification processes. A similar behavioral program has been observed in C. elegans during noxious heat exposure, which induces short-term heat avoidance followed by long-lasting cytoprotective adaptation and a gradual reduction in avoidance (Byrne Rodgers and Ryu, 2020).

The interaction of H2S with mitochondrial ETC is multifaceted, acting as an electron donor at low concentrations and becoming a potent toxicant at high levels (Szabo et al., 2014), in part by promoting superoxide generation through complex IV inhibition and reverse electron transport at complex I (Cooper and Brown, 2008; Jia et al., 2020; Khan et al., 1990; Nicholls and Kim, 1982; Romanelli-Cedrez et al., 2024). We propose that mitochondria play a dual role in H2S-evoked locomotory avoidance. On one hand, the mitochondrial ETC contributes to H2S detoxification and promotes adaptation. On the other hand, toxic levels of H2S remodel the ETC, leading to increased ROS production, which may serve as a trigger for the avoidance response. Supporting the idea that acute mitochondrial ROS generation initiates avoidance of high H2S levels, short-term rotenone exposure, known to promote mitochondrial ROS formation (Ochi et al., 2016; Ramsay and Singer, 1992; Zorov et al., 2014), substantially increases locomotory speed (Onukwufor et al., 2022). Meanwhile, the speed response to high H2S is fully suppressed by rotenone. This inhibition could result either from excessive mitochondrial ROS generated by rotenone, which may dampen the H2S-triggered ROS spike, or from direct complex I inhibition, which may disrupt other H2S signaling processes required to initiate avoidance. However, persistent mitochondrial ROS production appears to suppress high locomotory speed and inhibit responsiveness to H2S, as observed after 2 hr rotenone exposure, in mitochondrial ETC mutants, and in animals lacking all superoxide dismutases. One likely explanation is that mitochondrial ROS can activate a variety of stress-responsive pathways, including HIF-1, NRF2/SKN-1, and DAF-16/FOXO signaling (Lee et al., 2010; Lennicke and Cochemé, 2021; Patten et al., 2010), priming animals’ adaptation to prolonged stress rather than causing toxicity. This is supported by the observation that even though SOD-deficient animals do not display strong initial locomotory responses to H2S, they remain responsive to other stimuli after 30 min of H2S exposure, suggesting that high ROS levels do not compromise general viability or the H2S detoxification capacity. Therefore, we favor a model in which mitochondrial ROS exert a biphasic effect on H2S-induced avoidance, facilitating H2S avoidance under acute conditions and contributing to locomotory inhibition when it is chronically elevated. Overall, lack of a clear sensory machinery, the slow increase of locomotory speed in H2S (Figure 1D), the rotenone-evoked speed responses, and the strong modulation of H2S responses by mitochondrial ETC inhibition suggest that H2S may not be directly perceived by C. elegans. Instead, acute avoidance to H2S is likely initiated by ROS-induced toxicity.

In summary, this study unveils a novel behavior of C. elegans in their avoidance to H2S. The speed response to H2S is intricately shaped by environmental context, integrating inputs from other sensory cues such as food availability and O2 levels. Our findings suggest that H2S-induced locomotion arises from acute remodeling of mitochondrial ETC and associated ROS production, while highlighting the vital role of the HIF-1-induced detoxification pathways and iron homeostasis in protecting against H2S-induced mitochondrial toxicity. Further work is needed to validate this model and to elucidate the neural circuits mediating the behavioral response to ROS-induced toxicity.