C. elegans strains N2 (Bristol strain, wild-type) was provided by the Caenorhabditis Genetics Center (CGC), University of Minnesota, MN, United States. Strain XD321 aha-1(xd4) I was provided by the Mei Ding laboratory. This strain has a G/A substitution at the second splicing site, resulting in a decrease in the expression levels of aha-1 (Zhang et al., 2013). Strains VQ1310 qvIs8 [psur-5::luc::gfp + punc-122::RFP], VQ1071 qvEx295 [paha-1::luc::gfp] and VQ1324 qvEx361 [plin-42::luc::gfp::pest, pCFJ90] were previously generated in the laboratory. The XD321 strain was crossed with the VQ1310 qvIs8 strain, generating the VQ1722 aha-1(xd4) I; qvIs8 [psur-5::luc::gfp + punc-122::RFP] strain.

Transgenic nematodes were generated by standard microinjection techniques (Mello and Fire, 1995) through a collaboration with the laboratory of Dr. Claire Bénard (Université du Québec à Montréal, Canada). The integration of psur-5::luc::gfp was induced by UV radiation to generate qvIs8 (Mariol et al., 2013).

C. elegans strains were maintained on NGM medium (0.3% NaCl, 0.25% Peptone, 5 μg/mL cholesterol, 1 mmoL/L CaCl₂, mmol/l MgSO₄, 1.7% Agar in 25 mmoL/L of potassium phosphate buffer pH 6.0) with thick bacterial lawns of Escherichia coli HB101 strain under a dual cycle of light and temperature, Light/Dark (LD, ~150/0 μmol/m²/s) and Cold/Warm (CW, 18.5/20°C, Δ = 1.5°C ± 0.5°C) cycles 12:12 h. Light and temperature are known to be the main zeitgebers, and in this entrainment protocol ZT0 (9:00 AM) indicates the time of lights on and the cold phase onset. Circadian Time (CT) refers to a specific time in the free-running (FR) conditions (i.e., DD/WW cycles). Photo and thermal conditions were controlled with an I-291PF incubator (INGELAB, Argentina) and temperature was monitored using DS1921H-F5 iButton Thermochrons (Maxim Integrated, United States). Four cool white LED light strips (approx.150 μmol/m²/s) were used as the light source for the luminescence recordings. Nematode populations were synchronized to the same developmental stage by the chlorine method (Stiernagle, 2006).

Luminescence recording was carried out according to a protocol developed by the laboratory (Goya et al., 2016). Briefly, approximately 50 L4 hermaphrodite nematodes were manually selected for their higher GFP expression by picked under an SMZ100 stereomicroscope equipped with an epi-fluorescence attachment (Nikon) with a cool Multi-TK-LED light source (Tolket) to avoid warming of the plate. The nematode populations were washed once with M9 buffer (42 mM Na₂HPO₄, 22 mM KH₂PO₄, 85.5 mM NaCl, 1 mM MgSO₄) to remove all traces of bacteria and resuspended in 200 μL of luminescence medium: Leibovitz’s L-15 media without phenol red (Thermo Fisher Scientific) supplemented with 1X antibiotic–antimycotic (Thermo Fisher Scientific), 40 μM of 5-fluoro-2′-deoxyuridine (FUdR) to avoid new eclosions, 5 mg/mL of cholesterol, 10 μg/mL of tobramycin (Tobrabiotic, Denver Farma), 1 mM of D-luciferin (Gold Biotechnology) and 0.05% Triton X-100 to increase cuticle permeabilization. Nematode populations were then transferred to a white, flat-bottomed 96-well plate (Greiner), sealed with optical film (Microseal B PCR Plate Sealing Film, Biorad) and perforated to prevent condensation and allow oxygen exchange. Then, at ZT12 (21 h) of the same day, the plate was placed in a Berthold Centro LB 960 microplate luminometer (Berthold Technologies) stationed inside an incubator model G291PF (INGELAB, Argentina) to allow tight control of the light and temperature in each experiment. Microwin 2000 software 4.43 (Mikrotek 2 Laborsysteme) was programmed to leave the plate outside the luminometer after each recording to expose nematodes to the environmental cues. The luminescence of each well was integrated for 10 s every 30 min. Luminescence was monitored for 3 days under a dual 24 h LD/CW (CW, 15.5/17°C, Δ = 1.5 ± 0.5°C) cycle and then 3 days under FR (DD/WW; 17°C) conditions. The signal emitted by the nematodes was integrated for 10 s and recorded with an interval of 30 min.

Luminescence was sampled at 30 min intervals. Background noise was extracted from the raw data obtained from the luminometer. In all cases, the first 12 to 24 h of recording were removed due to accumulation of the luciferase enzyme. All raw data were analyzed using a Shiny app developed in the laboratory.¹ The raw data were detrended, smoothed and normalized to the initial maximum value of each sample and plotted using R. All data are shown as mean ± SEM of luminescence as indicated in the figures. In each case, the mean corresponds to a population of nematodes (50 nematodes). Subsequently, the period under LD/CW cycles and FR conditions was calculated from the population data using the Lomb-Scargle (LS) periodogram using the lomb R package (Van Dongen et al., 1999), within a range of 18 to 37 h and with an oversampling of 30. The acrophase (time at peak) and amplitude of each signal was estimated using the Cosinor method, by fitting a cosine waveform to the data using a non-linear least squares regression implemented using the nls function of base R and obtaining the R² of the fit. Any signal resulting from population analysis with a 24 h period and an R² adjustment ≥ 0.5 was considered “Synchronized” under cyclic training conditions. In the case of FR rhythms, any signal resulting from the analysis with a period range between 18 h and 37 h, and an R² adjustment ≥ 0.5 was considered “Circadian.” For statistical analyses, the GraphPad Prism 7 software was used. Statistical significance was set at alpha = 0.05. Final figures were generated using Biorender.²

Nematode populations (N2 strain) were synchronized to the same developmental stage by the chloride method (Stiernagle, 2006) and cultured overnight in a 50 mL Erlenmeyer flask with 3.5 mL of M9 buffe, 1X antibiotic-antimycotic (Thermo Fisher Scientific) and 10 μg/mL of tobramycin (Tobrabiotic, Denver Farma) at 110 rpm in LD/CW cycle (18.5/20°C). The next day, L1 larvae were placed on NGM plate and were grown for 48 h to the L4 stage under the same LD/CW cycle. Then, independent populations of approximately 4,000 nematodes at the L4 stage were washed with M9 buffer to remove the remains of bacteria and transferred to four 500-ml flasks with 130 mL of luminescence medium without D-luciferin (5 nematodes/10 μL). The nematodes then were cultured under LD/CW (15.5/17°C) cycle with agitation at 100 rpm for two additional days. Then they were released in FR conditions (DD/WW; 17°C) for the two remaining days of the assay. Four independent biological replicates (n = 4; 4,000 adult nematodes in each replicate) were collected every 4 h, starting at ZT1 (10 AM) in the LD/CW (15.5/17°C) cycle during the last day of entrainment and during the following 2 days of the FR condition (DD/WW; 17°C). Nematodes were centrifuged to discard the medium, washed, and frozen at −80°C in Trizol (Thermo Fisher Scientific). Total RNA was extracted from the samples using the Trizol method according to the manufacturer’s instructions. RNA solutions were quantified using NanoDrop1000 (Thermo Fisher Scientific), and their integrity was evaluated by electrophoresis. Two micrograms of total RNA and poly-T primers (20 nt long) (Thermo Fisher Scientific) were used for cDNA synthesis using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific). Gene amplification was performed on a QuantStudio 3 PCR instrument (Thermo Fisher Scientific), using 10 μL of final reaction volume containing 1 μL of cDNA as the template, 1 × of the SYBR Green PCR Master Mix 3.0 (PB-L), and the corresponding primers at a final concentration of 0.2–0.6 μM. The cDNA template was amplified in duplicate, with the following conditions: 95°C for 10 min, followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. Relative gene expression was calculated using the 2r^{− ΔΔCt} method, and Y45F10D.4 was the reference housekeeping gene. For pPCR analyses, the JTK_CYCLE algorithm implemented in the MetaCycle package (R) was used (p = 1; not statistically significant).

The primers used for amplification were:

Fw-aha-1: 5´-GTTCGTGTTTCGGAAGATGG-3′,

Rv-aha-1: 5´-TTCTCATCTGCTGGATGTGC-3′,

Fw-Y45f10D.4: 5´-GTCGCTTCAAATCAGTTCAGC-3′,

Rv-Y45f10D.4: 5´-GTTCTTGTCAAGTGATCCGACA-3′.

To assess a possible circadian expression in the expression of the aha-1 gene, we recorded luminescence rhythms from VQ1071 qvEx295 [paha-1::luc::gfp::pest] strain. Animals were entrained to a dual LD/CW cycle (18.5/20°C, Δ = 1.5°C) and the bioluminescent activity of the aha-1 promoter was measured thereafter in adult nematodes over three days in LD/CW (15.5/17°C, Δ = 1.5°C) cycles and then for three days under constant conditions (DD/WW, 17°C) (Figure 1A). We choose the 15.5/17°C cycle for recordings because 15.5°C is the minimal temperature allowed by the luminometer used and because the populations have a longer life span under this condition than at 18.5/20°C, resulting in a sustained luminescence signal for more days than at higher temperatures. Importantly, the temperature delta (1.5°C) is the same in both conditions. The VQ1071 strain exhibited a rhythm in the activity of the aha-1 promoter under cyclical and FR conditions, which increased during the nocturnal/warm phase and decreased during the cold phase (Figure 1B; Supplementary Figure S1). Under LD/CW and FR conditions, rhythmic adult populations showed a period of 23.76 ± 0.25 h and 24.88 ± 1.00 h, respectively (n = 14 mutant rhythmic, n total = 30, ~ 47% rhythmic) (Figure 1C; Supplementary Table S1). Approximately 32% of the rhythmic populations were synchronized following this entrainment protocol, as indicated by the acrophase distribution under cyclic conditions (LD/CW, light blue dots) and FR (DD/WW, pink dots) (Figure 1D). Supplementary Table S2 shows circadian period estimation by different methods, showing similar results.

To gain insights into the aha-1 expression pattern, we performed qPCRs experiments to quantify the mRNA levels for the aha-1 gene from bulk RNA extractions from adult worms. Samples were taken every 4 h for one day under cyclic conditions (LD/CW, 15.5/17°C) and for one day under constant conditions (DD/WW, 17°C), starting from the young adult stage. Although no circadian pattern for aha-1 gene was revealed (JTK_CYCLE, p = 1), we did detect the expression of gene throughout the experiment (Figure 1E). A daily variation was observed under LD/CW conditions, with peak expression during the diurnal phase; the expression levels of aha-1 decreased under constant conditions. It is still possible that orthologous clock genes do cycle in a small subset of cells or tissues in worms and that this get lost from bulk RNA extraction. Using published data single-cell RNA sequencing by CeNGEN (Hammarlund et al., 2018), which provides gene expression profiles of all C. elegans neurons in L4 nematodes, we found that the gene aha-1 is expressed in sensory neurons, interneurons, motor neurons, and pharyngeal neurons (Supplementary Figure S2A). Furthermore, using scRNA-sequencing data from a published database of gene expression in young adult and aging C. elegans (Ghaddar et al., 2023; Roux et al., 2023), we see that, the gene aha-1 is expressed in neurons and other cell types, in young adults and aging nematodes (Supplementary Figure S2B). Moreover, we examined the expression of aha-1 across different adult stages using the publicly available RNA-seq dataset from the C. elegans Aging Atlas.³ According to this dataset, the expression levels of aha-1 remain stable from day 1 to day 14 of adulthood in neuron as well as in other tissues (Supplementary Figure S2C). This suggests that the overall abundance of aha-1 transcripts does not change significantly during aging.

In C. elegans, per is conserved as lin-42 (Romanowski et al., 2014). Our laboratory previously showed that mutations of lin-42 generate a significantly longer endogenous period, suggesting a role for this gene in the nematode circadian clock, as in other organisms (Lamberti et al., 2024). We also examined luminescence rhythms of VQ1324 qvEx361 [aha-1::luc::gfp::pest, pCFJ90] strain under cyclic conditions (LD/CW, 15.5/17°C) followed by constant dark and temperature conditions (DD/WW, dark and 17°C). Interestingly, the luminescence rhythms for the lin-42 promoter were in antiphase to the bioluminescence rhythm of the aha-1 promoter under cyclic conditions (Figures 2A,B). Under FR conditions, rhythmic adult populations showed a period of 24.59 ± 1.14 h (VQ1324, n = 9 rhythmic, n total = 26, 34.6% rhythmic) (Figure 2C; Supplementary Table S1). We also used publicly available RNA-seq data from the C. elegans Aging Atlas (see text footnote 3) (Gao et al., 2024), and found that aha-1, lin-42, and kin-20 (another critical regulator of the adult nematode circadian clock through neuronal cells) are expressed in pharyngeal and sensory neurons (Supplementary Figure S2D). This supports the hypothesis of a potential interaction between aha-1 and lin-42. The specific cells involved in the circadian clock of C. elegans remain unidentified, but neurons are likely candidates, as observed in other organisms. Silencing candidate genes in neurons will be necessary to assess their potential role.

To examine whether the AHA-1 protein is a component of the nematode molecular clock, we used the luciferase reporter aha-1::luc::gfp::pest as a clock output, integrating the construct into the nematode genome by UV radiation (control strain VQ1310 qvls8, Supplementary Table S1). We then introduced the reporter into aha-1 mutant backgrounds by genetic crosses with this control strain (strain VQ1722 aha-1(xd4) I; qvIs8 [psur-5::luc::gfp + punc-122::RFP], Supplementary Table S1). Animals were entrained to a dual LD/CW cycle (18.5/20°C), and the bioluminescent activity of the sur-5 promoter was measured thereafter in adult nematodes over three days in LD/CW (15.5/17°C) cycles and then for three days under constant conditions (DD/WW, 17°C). The control and the aha-1(xd4) strains exhibited a sur-5 promoter rhythm under cyclical and FR conditions, thereby providing evidence that nematodes can be synchronized through a dual cycle of light and temperature (Figure 3A; Supplementary Figure S3). Under FR conditions, aha-1(xd4) nematodes showed a peak of luminescence during the subjective day, in antiphase to the control strain (Figure 3A). Both strains exhibited weak entrainment following this protocol, as evidenced by the acrophase shift in LD/CW and DD/WW conditions, suggesting that a masking mechanism is involved (Figure 3B). Although there was considerable high variability of circadian period across the rhythmic mutant populations, we found that aha-1(xd4) animals showed a significantly longer period than the control strain (27.14 ± 0.61 h, n = 36 mutant rhythmic, n total = 71, ~ 51% rhythmic vs. 24.24 ± 0.54 h, n = 20 control rhythmic, n total = 45, ~ 44% rhythmic) (Unpaired t-test, *p = 0,0256) (Figure 3C). These results suggest that aha-1 could modulate the period of sur-5-driven luminescent rhythms. To strengthen this finding, it would be necessary to evaluate whether a transgene of aha-1 can rescue the aberrant behavior observed in the aha-1(xd4) mutant when overexpressed from an extrachromosomal array under its native promoter.