Introduction

Sensory perception is fundamental to how organisms interact with their surroundings. Animals, including Caenorhabditis elegans, detect and respond to a wide range of environmental cues such as light and odorants. Despite having a compact nervous system of only 302 neurons, C. elegans exhibits sensory capabilities that allow it to effectively navigate and adapt to its environment (Bargmann, 2006; Cook et al., 2019). Odorants such as volatile organic compounds are detected via specialised olfactory receptor neurons. These neurons express specific receptors that bind to specific molecules and trigger distinct behavioural responses (Bargmann, 2006; Bargmann et al., 1993; Bargmann & Horvitz, 1991). Diacetyl, a volatile organic α-diketone found in C. elegans’ natural environment elicits opposing behaviours depending on its concentration. At low concentrations, diacetyl is sensed by the olfactory receptor ODR-10, promoting attraction (Itskovits et al., 2018; Sengupta et al., 1996; Zhang et al., 1997). In contrast, higher concentrations induce an avoidance response, thought to be mediated by the receptor SRI-14 (Taniguchi et al., 2014).

In addition to chemical cues, C. elegans also detects and responds to light, despite lacking eyes or classical photoreceptors. This light sensitivity is mediated by LITE-1, a member of the Gustatory Receptor family (Montell, 2009). LITE-1 was first identified through genetic screens for mutants defective in light avoidance behaviour (Edwards et al., 2008). Unlike canonical photoreceptors such as rhodopsins or opsins, LITE-1 shares little sequence similarity with known light sensing proteins (Edwards et al., 2008; Gong et al., 2016; Liu et al., 2010). Further underscoring its uncanonical nature, LITE-1 adopts an inverted membrane topology that is unlike classical G protein-coupled receptors (GPCRs) (Gong et al., 2016).

LITE-1 phototaxis behaviour is driven by multiple light responsive sensory neurons, including ASJ, ASH and ASK (Liu et al., 2010; Ward et al., 2008; Zhang et al., 2022). Signal transduction occurs via G protein signalling, upregulation of cGMP levels and activation of cyclic nucleotide-gated (CNG) channels, leading to a rapid avoidance response (Edwards et al., 2008; Liu et al., 2010; Ward et al., 2008). Recent studies suggest that LITE-1 may function as a light activated ion channel with a putative ligand binding pocket that is activated by ultraviolet and blue light via key tryptophan residues (Edwards et al., 2008; Gong et al., 2016; Hanson, Scholüke, et al., 2023; Ward et al., 2008).

Here we show that beyond its role in photoreception, LITE-1 is also required for the avoidance of high concentrations of diacetyl and that diacetyl induces muscle contraction in transgenic worms expressing LITE-1 in body-wall muscles. Our findings support the notion that LITE-1 functions as a multifunctional receptor involved in both light and chemical sensing.

Results

LITE-1 is required for diacetyl and blue light avoidance

A recent screen of a panel of worm strains that collectively contained knockouts of all non-essential GPCRs identified a triple mutant that failed to avoid high concentrations of diacetyl (Pu et al., 2023). One of the mutated genes was lite-1. We performed a chemotaxis assay to test single mutants of several loss-of-function alleles of lite-1 and found they all failed to avoid high concentrations of diacetyl (Fig. 1A and 1B) while remaining attracted to lower concentrations (Fig. 1C). As expected, these lite-1 mutants no longer respond to blue light stimulation (Fig. 1D) (Edwards et al., 2008).

Figure 1:

Chemosensory neurons ADL, ASK and ASH are involved in avoidance of diacetyl

To identify neurons involved in the response to high concentrations of diacetyl, we used calcium imaging to record from 11 pairs of sensory neurons in worms exposed to a pulse of diacetyl (Fig. 2A). In lite-1 mutants, the LITE-1-expressing sensory neurons ADL and ASK showed a clear defect in diacetyl response, whereas another LITE-1-expressing neuron ASH showed a normal response (compared to WT animals, Fig. 2B). The response of the other tested neurons was not significantly different between mutant and WT animals (Fig. 2B). The normal response to diacetyl in ASH in the absence of LITE-1 is likely due to its expression of SRI-14 which has previously been shown to respond weakly to high concentration of diacetyl (Taniguchi et al., 2014). We also recorded from unc-13 mutants which are impaired in synaptic activity and found little effect on the ADL and ASK responses (Fig. 2C) (Richmond et al., 1999). This is consistent with direct sensation in these neurons although neural signalling through gap junctions or via neuromodulation by other receptors could not be ruled out.

Figure 2:

LITE-1 is sufficient for diacetyl response in other cell types

To test whether LITE-1 is sufficient for a response to high diacetyl concentrations, we used a transgenic strain that expresses LITE-1 in body-wall muscles (Edwards et al., 2008). Worms ectopically expressing LITE-1 in body muscle cells evoke muscle contraction and shortening of body length in response to blue light stimulation (Edwards et al., 2008). When these worms are exposed to a 1:50 dilution of diacetyl, they showed a rapid and near complete paralysis whereas wild-type animals continue to swim (Fig. 3A and 3B). These worms also showed significant contraction in average body length, contracting by ∼11% after diacetyl exposure (Fig. 3C). These results suggest that LITE-1 is directly activated by diacetyl although it remains possible that it is a diacetyl metabolite that activates LITE-1 given that the full contraction develops over a minute time scale. Consistent with a ligand receptor relationship, molecular docking predicts low micromolar affinity of diacetyl binding to LITE-1 in the same binding pocket that was recently identified as a putative chromophore binding site (Fig. 3D) (Hanson, Scholüke, et al., 2023).

Figure 3:

2,3-pentanedione also interacts with LITE-1

We next tested whether lite-1 is required for avoidance of other high concentration odorants. Of the seven odorants tested, lite-1 mutants showed a defective response to 2,3-pentanedione, a chemical structurally related to diacetyl (Fig. 4A). The effect was weaker than that observed for diacetyl, with worms still avoiding high concentrations of 2,3-pentanedione but more weakly than wild-type animals. Supporting this observation, molecular docking predicts interaction between 2,3-pentanedione and LITE-1 putative binding pocket (Fig. 4B). Like diacetyl, 2,3-pentanedione also evokes paralysis and muscle contraction of worms expressing LITE-1 in muscle cells (Fig. 4C and 4D).

Figure 4:

Discussion

LITE-1 is best known for its role in light sensation in C. elegans, but how it senses light is not yet clear. In particular, an indirect mechanism in which light produces photoproducts that activate the channel has not been ruled out and a priori has always seemed plausible given that LITE-1 is similar to gustatory receptors that are known to sense chemicals (Montell, 2009). A direct diacetyl response would make a photoproduct-based mechanism more plausible. The identification of a second structurally related compound, 2,3-pentanedione may help in narrowing down photoproducts with similar moieties. Direct and photoproduct light sensing are not mutually exclusive. Based on LITE-1’s predicted structure and its absorption spectrum, it has recently been proposed that UV may be directly sensed while the blue light response may be mediated through a photoproduct or an unidentified chromophore (Hanson et al., 2023).

While our study primarily focused on chemosensory neurons, it remains possible that non-chemosensory neurons expressing LITE-1 may contribute to diacetyl detection or modulating the behavioural response. Indeed, a recent study demonstrated that the LITE-1-expressing interneuron AVG response to blue light stimulation by releasing the neuropeptide NLP-10 to mediate the escape behaviour (Dunkel et al., 2025). Nevertheless, phototransduction and chemotransduction appear to involve shared sensory neurons, such as ASK and ASH, suggesting a potential overlap in their underlaying signalling mechanisms (Zhang et al., 2022). It is possible that LITE-1 chemosensory response may rely on components necessary for LITE-1 photosensory function, such as G proteins (GOA-1, GPA-3), guanylate cyclase (DAF-11, ODR-1), CNG channels (TAX-2,4) and phosphodiesterase (PDE-1,2,5) (Liu et al., 2010; Ward et al., 2008). Furthermore, the use of lite-1 point mutants that affect specific LITE-1 function such as light sensing, channel gating or binding pocket could further elucidate LITE-1 mechanisms (Gong et al., 2016; Hanson, Scholüke, et al., 2023).

We have shown that LITE-1 is required for the avoidance of high concentrations of diacetyl and a related compound 2,3-pentanedione. Furthermore, we have shown that LITE-1 is sufficient to make muscle cells responsive to diacetyl and 2,3-pentanedione, consistent with a direct response. Diacetyl is an ecologically relevant molecule present in C. elegans’ natural environment that has been hypothesised to be involved in prey-finding since low concentrations produced by lactic acid bacteria on rotting fruit is attractive (Choi et al., 2016). Avoidance of high concentrations of diacetyl may therefore also be ecologically relevant. In this case, LITE-1 has likely evolved to be a multimodal receptor of aversive stimuli akin to other multimodal receptors such as TRPV1, which responds to heat, capsaicin, and acidic pH (Zhang et al., 2023).

Material and methods

Bacterial and worm strains maintenance

The E. coil wild-type isolate OP50 was sourced from Caenorhabditis Genetics Center (CGC) and maintained on lysogeny broth (LB) agar under standard laboratory conditions. The following worm strains were used in this study, N2; wild-type isolate (CGC), CHS1610; lite-1(yum2880) (Pu et al., 2023), KG1180; lite-1(ce314) (Edwards et al., 2008), KG1272; cels37[myo-3_prom::lite-1::GFP] (Edwards et al., 2008), TQ8245; lite-1(xu492) (Zhang et al., 2020), ZAS280; In[osm-6::GCaMP3, osm-6::ceNLS-mCherry-2xSV40NLS] (Iwanir et al., 2019), ZAS371; unc-13(s69) In[osm-6::GCaMP3, osm-6::ceNLS-mCherry-2xSV40NLS] (Bokman, Kalij, et al., 2024; Bokman, Pritz, et al., 2024), ZAS526; lite-1(ce314) In[osm-6::GCaMP3, osm-6::ceNLS-mCherry-2xSV40NLS], were cultured on Nematode Growth Medium seeded with E. coli (OP50) as previously described (Brenner, 1974). Worm synchronization was performed by bleaching unsynchronised gravid adults (Barlow, 2019).

Chemotaxis assay

The chemotaxis assays were conducted at 20°C in 9 cm plates filled with 10 ml of agar (1.7% agar, 1 mM CaCl₂, 1 mM MgSO₄ and 25 mM K₂HPO₄ pH 6). Two parallel lines, 1 cm apart, were drawn on the middle of the plates which define the area to place the animals. Two odorant spots were designated on one side of the plate, while two control spots were positioned on the opposite side (Fig. 1A). 1 μl of 1 M NaN₃ was added to all four spots to immobilise the animals. Different volumes of each odorant: diacetyl (5 μl), 2,4,5-trimethylthiazole (7.5 μl), benzaldehyde (2.5 μl), isoamyl alcohol (7.5 μl), 2,3-pentanedione (10 μl), 2-nonanone (1 μl), 1-octanol (2.5 μl), 2-butanone (10 μl) and equal volume of ethanol (control) were spotted onto the respective dots. Synchronized day-one adults were washed 3 times in chemotaxis buffer (1 mM CaCl₂, 1 mM MgSO₄ and 25 mM K₂HPO₄ pH 6) before being transferred onto the centre of the assay plate where the assay will last for 1 hr at 20 °C. The chemotaxis plates were then stored at 4 °C before counting. The chemotaxis index was calculated as (number of animals on the odorant side – number of animals on the ethanol side) / (sum of all animals on both sides).

Paralysis assay

The paralysis assays were conducted at 20°C in 6-well plates with 4 ml of agar (1.7% agar, 1 mM CaCl₂, 1 mM MgSO₄ and 25 mM K₂HPO₄ pH 6). Synchronized day-one adults were washed 3 times in chemotaxis buffer (1 mM CaCl₂, 1 mM MgSO₄ and 25 mM K₂HPO₄ pH 6) before being transferred onto the centre of the 6-well plate. 1 ml of chemotaxis buffer containing diacetyl (1:50) was then added into each well and videos were immediately acquired at 25 frames per second using a shutter speed of 25 ms and a resolution of 12.4 μm/pixel.

Blue light stimulation assay

The blue light stimulation assays were conducted at 20°C in 24-well plates containing agar (1.7% agar, 1 mM CaCl₂, 1 mM MgSO₄ and 25 mM K₂HPO₄ pH 6). Synchronized day-one adults were transferred onto the centre of the 24-well plate pre-seeded with OP50. The plates were then transferred onto the multi-camera tracker for 30 min to allow acclimatization to the tracker room before imaging. Videos were acquired at 25 frames per second using a shutter speed of 25 ms and a resolution of 12.4 μm/pixel. Videos were taken in the following order: 300 s pre-stimulus, blue light stimulus with one 10 s pulse of blue light at 60 s and a 100 s post-stimulus recording. Videos were then processed with Tierpsy Tracker as previously described (Barlow et al., 2022; Javer et al., 2018).

Calcium imaging

Young adult worms were inserted into a microfluidic chip and anesthetized with 10 mM levamisole and allowed to habituate for 10 mins as previously described (Chronis et al., 2007). Recordings began with 1 min of light habituation, after which the stimulus was presented for 1 min The stimulus used was 1:5 diacetyl in CTX buffer (5 mM KH₂PO₄/K₂HPO₄ pH 6, 1 mM CaCl₂ and 1 mM MgSO₄). Recordings were made using a Nikon A1R + confocal laser scanning microscope with a water immersion × 40 (1.15NA) objective controlled by the Nikon NIS-elements software. Z-axis resolution was 0.5-0.8 μm. Acquisition rate was ∼1.5 volumes/second, interpolated to a final framerate of 2 Hz.

Neuron identification and signal extraction

Neuron segmentation and tracking was done on the nuclear mCherry signal using an algorithm developed by (Toyoshima et al., 2016). Neurons were identified visually based on anatomical positions. Neurons that could not be unambiguously identified were removed from analysis. Calcium readings were taken from within 0.9 of the radius of the segmented nuclear sphere. Signal intensity was normalized by baseline activity, defined as the lowest point of a 10-frame running average. All data analyses were performed using in-house MATLAB scripts (Pritz et al., 2023).

Molecular docking

Molecular docking was performed on the LITE-1 tetramer structure using DynamicBind (Hanson, Scholuke, et al., 2023; Lu et al., 2024). Additional rounds of docking on top ranked poses were performed with Gnina (McNutt et al., 2021).

Acknowledgements

We thank Na Yu for technical assistance and Denise Walker, Bill Schafer, and Alexander Gottschalk for providing strains. This work was funded by the Medical Research Council (grant MC-A658-5TY30 to AEXB) and the Israel Science Foundation (grant 1939/23 to AZ). Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

Additional information

Funding

Medical Research Council (MC-A658-5TY30)

Israel Science Foundation (1939/23)