Chemical synchronization using household bleach can affect development, behavior and lifespan in Caenorhabditis elegans

ABSTRACT

Caenorhabditis elegans, a nematode genetic model, is often synchronized in experiments to accurately observe its unbiased phenotypes. Chemical synchronization, simple bleaching of gravid worms, has been widely used to obtain eggs in bulk because the eggshell is extremely resistant to harsh conditions. While those eggs are considered to preserve their natural properties and are allowed to hatch to obtain synchronized worms, it has not been fully investigated whether chemical synchronization influences embryonic and post-embryonic traits. In this study, we compared some phenotypes observed in worms collected by picking laid eggs and by chemical synchronization. Chemical synchronization decreased hatching and developmental rates and, in some conditions, affected morphological traits, such as body length and head size, and brood size. In contrast, chemical synchronization did not affect the developmental potential of early- or later-stage eggs, or chemosensory behaviors. While the lifespan of wild-type worms did not change, the daf-16 loss-of-function mutant with an extremely short lifespan showed much variation when chemically synchronized. This study clarifies the impacts of chemical synchronization of worms and provides some critical considerations for its use.

Introduction

Caenorhabditis elegans has become the go-to model organism for genetics, development, neurobiology, and aging, mainly due to its quantifiable developmental and behavioral traits, a range of genetic toolkits, and use of microfluidics and omics technology (Girard et al. 2007; Corsi et al. 2015). A critical factor to consider for C. elegans experiments is the fact that the characteristics of C. elegans change in accordance with worm age and developmental state. Specifically, different developmental stages of animals could also yield different results with the same external stimulation. As noted, even small age variation within several hours could have an effect on the heat shock response in the young adult stage (Labbadia and Morimoto 2015). To use C. elegans in experiments, it's important to have the worms grow at the same developmental stage.

Egg-laying synchronization is achieved by collecting eggs after a short period, usually ranging from 30 min to 2 h, during which adult worms are allowed to lay eggs. This synchronization method is a ‘natural’ way to observe worms. An embryo of C. elegans is protected inside a multilayered eggshell, with the chitin layer being the last one that specifically provides the mechanical strength and shield from chemicals (Olson et al. 2012). As soon as gravid adult worms are immersed in the ‘bleaching solution’, a mixture of sodium hypochlorite, a major gradient of household bleach, and sodium hydroxide, the adult body mass starts to get decomposed, and the eggs, released from the uterus. The collected eggs usually hatch and produce larvae that normally develop and behave when cultivated under optimal conditions. This chemical synchronization has been widely used because it makes it possible to collect a large number of eggs quickly and at the same time to start a clean culture by decontaminating them (Stiernagle 2006). Different formulations of the bleaching solution, with the usage of different concentrations and treatment durations, effectively yield eggs that appear not to differ regarding their observed phenotypes (Porta-de-la-Riva et al. 2012). Along with growing interest in artificial intelligence (AI) assay automation, microfluidic devices for handling C. elegans use the prevailing method of chemical synchronization to obtain more eggs in less time (Midkiff and San-Miguel 2019).

Despite its convenience and the flexible way to adjust, recent studies suggest that chemical synchronization can have an effect on the egg of C. elegans. The finding of a decrease in rigidity of the eggshell after chemical synchronization indicates that the chitin layer could be damaged by the treatment (Krenger et al. 2020). In fact, the eggs collected through chemical synchronization were shown to have a decreased viability rate in a dose-dependent manner (Verdu et al. 2022). In contrast, the lifespans of N2 wild-type and long-lived isp-1(qm150) at 20°C are not changed by chemical synchronization (Tamez Gonzalez et al. 2024). Nevertheless, whether the chemical synchronization changes other parameters like developmental rate, behavioral traits, or lifespan at different temperatures (like 18 and 25°C) remains widely unexplored.

In this study, we synchronized C. elegans through egg-laying or chemical synchronization, then compared embryonic and post-developmental traits in animals collected from six different synchronization conditions: egg-laying control and weak, moderate, or strong bleaching solution for 5 s or 5 min. The chemical synchronization reduced the hatch rate, slowed down the growth, and in some cases, changed morphological traits like body and head size, and most of all, decreased brood size. Nevertheless, it did not affect the chemotaxis behaviors toward benzaldehyde, isoamyl alcohol, 2-butanone, diacetyl, cupric chloride, or glycerol-induced hyperosmolarity. Chemical synchronization, in general, did not impact the lifespan of N2 wild-type worms, but it generated variations in the lifespan of the daf-16 loss-of-function mutant worms, which live longer than wild types. Our study illustrates that chemical synchronization may play a role in increasing the phenotypic variation in the population, thereby leading to greater fluctuation of individual replicates in experiments. As a result, the exact configuration and unchanging bleaching conditions are necessary for achieving reproducibility and fidelity in the research.

Materials and methods

Materials

All reagents were purchased from Sigma-Aldrich, unless otherwise mentioned.

Maintenance of worms

The wild-type Bristol N2 and daf-16 (mu86) were obtained from the Caenorhabditis Genetic Center (Minneapolis, MN) and maintained as previously described (Brenner 1974).

Synchronization

We prepared the plates containing gravid worms mostly at the day-2 adult stage. For egg-laying synchronization, 40 hermaphrodites were transferred to a fresh plate and allowed to lay eggs for 2 h. This egg-laying synchronization was performed in parallel with chemical synchronization as a control. For chemical synchronization, we used three bleaching solution concentrations of ‘Weak’, ‘Moderate’, and ‘Strong’ for 5 s or 5 min with occasional brief mixing (Figure 1A). Day-2 adult-stage gravid animals were washed with M9 buffer, and then each bleaching solution was added to the final concentration. After washing, eggs were placed on OP50-seeded NGM using a Pasteur pipette and incubated at 20 °C. L4 animals from the egg-laying or chemical synchronization were transferred to new plates for further examination. To collect staged eggs from non-bleached worms, the uterus of day-2-adult-stage N2 wild-type animals was punctured with a microinjection needle and transferred released eggs with an eyelash taped to a toothpick.

Figure 1.

Hatch rates are reduced by chemical synchronization. (A) The chemical synchronization process. Day-2 adult worms were treated with weak (W), moderate (M), or strong (S) bleaching solutions for either 5 s (5s) or 5 min (5 m). After bleaching, eggs were placed onto OP50-seeded NGM plates. For further experiments, early (E)- and later (L)-stage eggs were collected. (B) Hatch rates of six different combinations of conditions are plotted (Ctrl, control). Three independent experiments were performed. n = 60–207 per experiment. (C) Hatch rates of pooled data. To evaluate the effects of treatment duration and bleaching solution concentration, data were pooled as described in the ‘Statistics’ section of the Materials and Methods. (B, C) Error bars represent SEMs. * < 0.01, ** < 0.005, *** < 0.001, **** < 0.0001. Two-way ANOVA with Holm-Sidak multiple comparisons.

Hatch rate and developmental rate

Images of eggs on plates were taken right after synchronization, and the number of eggs in each image was counted. 19 h later, the eggs that remained were scored. Images of worms in plates were taken at 24, 48, and 72 h, and the number of worms at each developmental stage was counted. A Zeiss Image A1 microscope equipped with a Zeiss AxioCam MRm camera was used for imaging.

Morphological characteristics

Day-4 adult-stage animals were mounted on the 2% agarose pad and immobilized with levamisole, and then images were taken under 100x magnification for the whole body and 400x for the head region. Zeiss Axiovision software was used to measure the body length, body width, pharynx length, and head size. For body length, we measured the curved midline from the tip of the head to the tip of the tail. For body width, we measured the ventral-to-dorsal width at the vulva. The pharynx length refers to the length of the curved midline that follows the pharyngeal tract from the tip of the pharynx to the terminal bulb. The head size is the length of the curved midline from the tip of the head to the terminal bulb (Cho et al. 2021).

Chemosensory behaviors

Chemotaxis assay was modified from the previous protocol (Margie et al. 2013). Benzaldehyde, isoamyl alcohol, 2-butanone, and diacetyl were diluted in ethanol, whereas copper(II) chloride dihydrate or glycerol was diluted in M13 buffer. We performed chemotaxis and a drop assay a day after synchronized animals reached the L4 stage. Chemotaxis assay plates were prepared as previously described (C. I. Bargmann and Horvitz 1991). Four points were established at the vertices of a 5 cm × 5 cm square centered on the plate. Two test (T) and two control (C) sites were designated on the top of the four points, alternating between each quadrant. A circle with a diameter of 2 cm was drawn at the center of the plate. 100–200 animals were placed at the center of the chemotaxis plate. 3 μL of 1 M sodium azide was applied at the four points, as well as 3 μL of the test reagent or ethanol, at the two T points and two C points, respectively. After closing the lid, the plates were placed upside down inside the 20 °C incubator. An hour later, they were transferred to the 4 °C fridge until counting. The animals inside the inner small circle were censored, and the number of animals in each quadrant was counted to calculate the chemotaxis index:

$$\mathrm{Chemotaxis}\mathrm{Index}\left(C.I\right)={\displaystyle \frac{\left(\mathrm{The}\mathrm{number}\mathrm{of}\mathrm{animals}\mathrm{in}T\mathrm{quadrants}\right)-\left(\mathrm{The}\mathrm{number}\mathrm{of}\mathrm{animals}\mathrm{in}C\mathrm{quadrants}\right)}{\mathrm{Total}\mathrm{number}\mathrm{of}\mathrm{animals}\mathrm{in}\mathrm{the}\mathrm{plate}}}$$

For a drop assay, we washed synchronized day-one-adult worms with M13 buffer three times, placed them on a chemotaxis plate, and allowed them to crawl for 5 min. Then, we dropped copper or glycerol solution onto the tail of the forward-moving animal. The number of animals that moved backward as far as half their body length was counted and used to calculate the avoidance index:

$$\mathrm{Avoidance}\mathrm{Index}\left(A.I\right)={\displaystyle \frac{\mathrm{The}\mathrm{number}\mathrm{of}a\mathrm{n}\mathrm{imals}\mathrm{that}\mathrm{performed}a\mathrm{full}\mathrm{backward}\mathrm{movement}}{\mathrm{Total}\mathrm{number}\mathrm{of}\mathrm{animals}\mathrm{exposed}\mathrm{to}\mathrm{reagent}}}$$

Lifespan assay

For each experiment, two plates containing 50–60 L4-stage animals were prepared and incubated at 16, 20, or 25°C. Animals were scored as dead when they did not respond with head swaying or pharyngeal pumping to a gentle head touch. Bag of worms, worms with ruptured organs, and worms burrowing inside NGM or crawling up the side of plates were censored.

Brood size

Worms after synchronization were transferred to a new plate daily until they stopped laying eggs. The number of hatched animals was counted.

Statistics

To examine any effect of treatment duration, the concentration of bleaching solution, and chemical synchronization in general, we pooled data depending on treatment duration (e.g. ‘Weak, 5 s’, ‘Moderate, 5 s’, and ‘Strong, 5 s’ into ‘5 s’) or on concentration of bleaching solution (e.g. ‘Weak, 5 s’ and ‘Weak, 5 min’ into ‘Weak’). To evaluate the overall effect of the chemical synchronization, data from six chemical synchronization conditions were pooled into a new group, designated as ‘Chemical Synchronization’. Prism 8 from GraphPad was used for statistical tests and plotting. For the hatch rate, two-way ANOVA with Holm-Sidak multiple comparisons was performed. To compare the developmental rate between the control and the chemical synchronization, we used a chi-square test with Bonferroni correction. For the morphological characteristics, brood size, and pharyngeal pump rate, Brown-Forsythe and Welch ANOVA with Games-Howell multiple comparisons tests were performed, except for the comparison between control and data pooled across all chemical synchronizations, for which Welch’s t-test was used. For morphological characteristics of full-grown worms from early– and later-stage eggs, Brown-Forsythe and Welch ANOVA with Games-Howell multiple comparisons test were performed. For the chemotaxis and drop assay, we used two-way ANOVA with Tukey multiple comparisons test. For the lifespan assay, the log-rank test with Bonferroni correction was performed. We indicated significance levels: * < 0.01, ** < 0.005, *** < 0.001, **** < 0.0001.

Results

The hatch rate of early-stage eggs was significantly reduced by chemical synchronization

A variety of combinations of bleaching solutions and treatment durations are widely used without much distinction, and the eggs collected by various protocols of chemical synchronization are generally assumed indistinguishable from laid eggs. However, as different concentrations of bleaching solution affect the hatch rate of the collected eggs differently (Verdu et al. 2022), we set out to investigate the effect of chemical synchronization on widely observed phenotypes, such as brood size, developmental rate, chemosensory behaviors, and lifespan. We used three bleaching solutions and defined them as ‘Weak’, ‘Moderate’, and ‘Strong’, based on the concentrations of sodium hypochlorite (NaClO) and sodium hydroxide (NaOH) (Figure 1A) (Porta-de-la-Riva et al. 2012). Most chemical synchronization protocols do not specify a fixed treatment duration, but instead suggest stopping bleaching treatment when animal debris is no longer visible. To ensure reproducibility, we treated eggs with chemical synchronization for two fixed durations, 5 s or 5 min. Therefore, a total of six chemical synchronization conditions were used in parallel, with egg-laying synchronization as a control (Figure 1A).

It has been reported that the treatment with bleaching solution can reduce egg viability (Verdu et al. 2022). Eggs collected from chemical synchronization range from newly fertilized ones to those containing embryos. We thus investigated the hatch rates of early- and late-stage eggs following chemical synchronization. We selected 1– to 32-cell-stage eggs as ‘early-stage’ eggs, and freshly laid eggs containing bean- or comma-stage embryos as ‘later-stage’ eggs. For controls, we collected early- and later-stage eggs from gravid day-two adult-stage animals by tearing their uterus with a needle. The difference in hatch rate between early- and later-stage eggs of controls was less than 5% and not statistically significant (Figure 1B). In case of chemical synchronization, however, the hatch rates of early-stage eggs from six chemical synchronization conditions were 30.5–65% lower than those of later-stage eggs (Figure 1B). To examine the effects of treatment duration, bleaching solution concentration, or general chemical synchronization, we pooled data depending on treatment duration, bleaching solution concentration, or all six conditions. The reduced hatch rate of early-stage eggs compared with later-stage eggs remained evident. Only 47.6–56.8% of the early-stage eggs hatched from the chemical synchronization, while 95.3% hatched from laid eggs (Figure 1C). These results suggest that chemical synchronization can reduce the hatch rate of eggs, particularly those at early stages.

Because chemical synchronization can reduce hatch rate, we examined whether it affects worm development and growth. We collected eggs from chemical synchronization, counted the number of worms at each stage after 24, 48, and 72 h, and compared these counts with those from eggs laid within a two-hour window (Figure 2). The development of worms from the ‘Weak, 5 sec’, ‘Moderate, 5 sec’, and ‘Strong, 5 sec’ treatments began to be delayed at 24 h (Figure 2A). The developmental delay persisted and was finally observed across all chemical synchronization conditions at 72 h (Figures 2B and C). Notably, some eggs from all chemical synchronization conditions hatched very late, and L1 animals (up to 1%) were still observed at 48 h, a pattern never seen in the egg-laid population. We also observed that some eggs from chemical synchronization would take 2–3 days to hatch.

Figure 2.

Chemical synchronization delays development. Developmental stages were assessed at 24 (A, D, G, and J), 48 (B, E, H and K), and 72 h (C, F, I and L) following chemical synchronization using weak (W), moderate (M), or strong (S) bleaching solutions for 5 s (5s) or 5 min (5 m). (A–C) Developmental distributions of six different combinations of conditions. n = 454–1306. (D–L) Developmental distributions of pooled data. The data are pooled by treatment duration (D–F), bleaching solution concentration (G–I), and chemical synchronization (CS) treatment (J–L). See ‘Statistics’ in Materials and Methods for further details. (A–L) * < 0.01 ** < 0.005 *** < 0.001 **** < 0.0001. chi-square test with Bonferroni correction. The asterisks above the bar indicate statistical significance relative to the control.

Research results are often presented from pooled data from various protocols of chemical synchronization performed by multiple experimenters, also for blinding purposes. When data were pooled depending on treatment duration or bleaching solution concentration, delayed development was evident (Figure 2D–I). Interestingly, the distributions of developmental stage between 5 s and 5 min differed significantly (Figure 2D–F). In addition, pooled data of all chemical synchronizations also showed developmental delays (Figure 2J–L). Altogether, these results indicate that chemical synchronization can delay development, and late hatching may contribute.

Chemical synchronization can reduce the growth potential of the collected animals

C. elegans has a simple body structure and has been utilized to reveal genes regulating organismal development and growth (Tuck 2014; Cho et al. 2021). Also, external factors, such as diet and environment, can affect the genetically planned morphology of worms. Chronic embryonic exposure to 10% ethanol not only delayed embryonic development but also decreased the mean body length of day 1-, 2-, and 3 old animals (Lin et al. 2013). Therefore, we investigated whether chemical synchronization also causes morphological changes in worms. As previously described (Cho et al. 2021), we measured body length, body width, pharynx length, and head size of chemically synchronized animals at the day 4 adult stage. Then we compared these measurements with those of egg-laid controls. The body length tended to be shorter and the body width thicker in animals obtained through chemical synchronization with stronger or longer treatments (Figure 3A–D). The pharynx and head were also sensitive to longer or stronger chemical synchronization, getting shorter (Figure 3E and F).

Figure 3.

Morphological traits can be affected by chemical synchronization. (A, C, E, and G) Whisker plot of morphological characteristics of day 4 adult-stage animals chemically synchronized using weak (W), moderate (M), or strong (S) bleaching solutions for 5 s (5s) or 5 min (5 m). n = 76–124. (B, D, F, and H) Data were pooled as described in the ‘Statistics’ section in the Materials and Methods for treatment durations, bleaching solutions, and chemical synchronization (CS) regardless of duration or solution. The body length (A, B), body width (C, D), pharynx length (E, F), and head size (G, H) were measured as described in the Materials and Methods. (A–H) * or #<0.01, ** or ##<0.005, *** or ###<0.001, **** or ####<0.0001. Brown-Forsythe and Welch ANOVA with Games-Howell’s multiple comparisons test (*), and Welch’s t-test (#). The asterisks and hashtags above the whisker indicate statistical significance relative to the control.

The investigation of the developmental characteristics of animals collected from chemical synchronization revealed some notable results. First, the morphological traits of animals collected from 5-second treatment, regardless of bleaching solution concentration, resembled those of animals from egg-laying synchronization (Figure 3A, C, E, and G). Second, bleaching for 5 min yielded day-4 adult worms with shorter pharynx lengths than those of egg-laid animals (Figure 3E). Lastly, for 5 min, the concentration of the bleaching solution affected the body length, width, and head size of the day-4 adult animals non-uniformly (Figure 3A, C, and G). The magnitude of these effects decreased as the data were pooled, although they remained statistically significant (Figure 3B, D, and H).

We first showed that the hatch rate of early-stage eggs was significantly lower than that of later-stage eggs (Figure 1B and C). The assembly of the C. elegans eggshell finishes by anaphase II (Olson et al. 2012), supporting the reinforcement or modification of the eggshell. Therefore, we hypothesized that, as early-stage eggs are more sensitive to chemical synchronization, surviving early-stage embryos are less competent than later-stage embryos, resulting in different developmental potentials that contribute to altered morphological characteristics in chemically synchronized animals (Figure 3). We measured and compared the sizes of the body length, width, pharynx length, and head size of the day-4 adult-stage animals of early- and later-stage embryos from chemical synchronization and laid eggs. We first noticed that they were not significantly different from controls. Moreover, in the same chemical synchronization conditions, the developmental potentials of early- and later-stage eggs were not significantly different, either (Figure 4A, C, E, and G). No difference in developmental potential between early- and later-stage eggs persisted in the pooled analyses, further confirming our observation that chemical synchronization does not influence the developmental potential (Figure 4B, D, F, and H).

Figure 4.

Morphological change by chemical synchronization is independent from egg stage. Morphological traits were measured in adult animals hatched from early-stage (E) and later-stage (L) eggs obtained through chemical synchronization at three concentrations (W, weak; M, moderate; S, strong) for 5 s (5s) or 5 min (5 m). (A, C, E, and G) Whisker plot of morphological characteristics of day 4 adult worms of early-stage or later-stage eggs. At least three independent experiments were performed, with a total number of worms observed ranging from n = 21–112 per condition. (B, D, F, and H) Data were pooled as described in the ‘Statistics’ section in the Materials and Methods for treatment durations, bleaching solutions, and chemical synchronization (CS), regardless of duration or solution. The body length (A, B), body width (C, D), pharynx length (E, F), and head size (G, H) were measured as described in the Materials and Methods. (A–H) ns > 0.01. Brown-Forsythe and Welch ANOVA with Games-Howell’s multiple comparisons test.

5 min of chemical synchronization resulted in shorter pharynx length than controls (Figure 3E and F). We therefore asked whether pharyngeal pumping changes after chemical synchronization. None of the six chemical synchronization conditions affected the pharyngeal pumping rate (Supplementary Data S1A). This was also the case in the pooled data (Supplementary Data S1B). These results indicate that a shortened pharynx through chemical synchronization still properly functions.

Brood size of C. elegans at 20°C is reduced after harsh chemical synchronization

C. elegans hermaphrodites can lay 200–300 eggs through self-fertilization, and the large brood size makes it a useful tool for toxicological testing (Boyd et al. 2010; Hunt 2017). Exposure of chemically synchronized C. elegans eggs to a glyphosate-containing herbicide decreases egg viability and the number of offspring laid for 24 h (McVey et al. 2016). We therefore asked whether chemical synchronization influences the reproduction of C. elegans. We measured the brood size of the animals collected from egg-laying and chemical synchronization. Five out of six chemical synchronization conditions yielded animals with similar brood sizes to the control. However, the animals from ‘Strong, 5 min’ condition produced much smaller number of offspring than the control (Figure 5A). In the pooled data, 5 min of chemical synchronization yielded animals with a mean brood size 12.9% smaller than the control. Moreover, the brood size of chemically synchronized animals was significantly different from that of the control (Figure 5B). This result implies that harsh conditions such as ‘Strong, 5 min’ can affect the reproductive competence of C. elegans.

Figure 5.

Chemical synchronization can reduce brood sizes. (A) Dot plot of worm brood size from egg-laying (Ctrl, Control) and chemical synchronization with weak (W), moderate (M), or strong (S) bleaching solutions for 5 s (5s) or 5 min (5 m). 8–12 independent experiments were performed, with a total of 48–120 worms observed per condition. (B) Dot plot of pooled brood size. Data were pooled as described in the ‘Statistics’ section of the Materials and Methods for treatment durations, bleaching solutions, and chemical synchronization (CS), regardless of duration or solution. (A, B) Error bars represent SEMs. * or #<0.01, ** or ##<0.005, *** or ###<0.001, **** or ####<0.0001. Brown-Forsythe and Welch ANOVA with Games-Howell’s multiple comparisons test (asterisk, *), and Welch’s t test (hashtag, #). The asterisks and hashtags above the bar indicate statistical significance relative to the control.

Animals collected from chemical synchronization behave normally to benzaldehyde, isoamyl alcohol, 2-butanone, diacetyl, copper ions, and glycerol.

Exposure to benzaldehyde or isoamyl alcohol during the L1 larval stage increases the sensitivity toward moderately attractive concentrations of benzaldehyde or isoamyl alcohol, respectively (Remy and Hobert 2005). Adult worms that were exposed to pathogenic bacteria during the L1 larval stage persistently avoided the bacterial odors (Jin et al. 2016). This early-stage exposure-based memory, called olfactory imprinting, can alter the behavior of adult animals. Also, an alkaline pH below 10 is attractive to C. elegans (Ward 1973; Murayama et al. 2013), while a pH above 10 repels it (Sassa et al. 2013). Chemical synchronization has been widely used in chemotaxis assays, too. We showed that some chemical synchronization reduced the size of the pharynx, around which many sensory neurons are located (Figure 3E) (Cornelia I. Bargmann 2006; C. I. Bargmann et al. 1993; C. I. Bargmann and Horvitz 1991). Therefore, we asked if chemical synchronization affects the chemosensory behavior of C. elegans.

AWA and AWC amphid neurons sense various volatile odors (C. I. Bargmann et al. 1993). First, we tested benzaldehyde, isoamyl alcohol, and 2-butanone, sensed by AWC neurons. Although we initially observed substantial variation, especially in experiments with isoamyl alcohol, all six chemical synchronization conditions yielded animals that were statistically indistinguishable from egg-laid control animals (Figure 6A, C, and E). This similarity between egg-laying and chemical synchronization persisted in the pooled data (Figures 6B, D, and F). We next tested diacetyl, an AWA-sensed volatile chemical. We found no statistically significant differences between the egg-laid and the six chemical synchronization conditions at every tested diacetyl concentration, as well as in the pooled data (Supplementary Data S2). Finally, we asked if chemical synchronization influences the avoidance response to copper ions and hyperosmolarity induced by glycerol, both of which are sensed by ASH neurons. There was no significant difference observed between the egg-laid and each of the six chemical synchronization conditions (Supplementary Data S3). Taken together, these results suggest that chemical synchronization does not affect the chemotactic responses to benzaldehyde, isoamyl alcohol, 2-butanone, diacetyl, copper ions, and hyperosmolarity.

Figure 6.

Chemical synchronization does not affect chemotaxis against benzaldehyde, isoamyl alcohol, or 2-butanone. (A, C, and E) Dot plot of the chemotaxis index of day 1 adult worms that were egg-laid (Ctrl; control) or chemically synchronized using weak (W), moderate (M), or strong (S) bleaching solutions for 5 s (5s) or 5 min (5 m). N = 3–9 independent experiments for benzaldehyde, N = 7–16 for isoamyl alcohol, and N = 3–8 for 2-butanone. (B, D, and F) Data were pooled as described in the ‘Statistics’ section in the Materials and Methods for treatment durations, bleaching solutions, and chemical synchronization (CS), regardless of duration or solution. (A-F) Error bars represent SEMs. ns > 0.01 for all comparisons from two-way ANOVA with Tukey multiple comparisons test.

The lifespan of wild-type N2 at 16, 20, or 25°C is not affected by chemical synchronization

The lifespan of C. elegans is regulated by multiple genetic and environmental factors (Uno and Nishida 2016). Chemical synchronization is the preferred method for measuring C. elegans lifespan because it is easy to use to collect large numbers of worms (Amrit et al. 2014). It is assumed that there is no difference in lifespan between worms hatched from laid eggs and chemically synchronized worms. One study showed that the lifespans of wild-type N2 and the long-lived mutant isp-20(qm150) at 20°C were not affected by chemical synchronization; in this condition, 5-Fluoro-2’-deoxyuridine (FUdR) was used to inhibit offspring production (Tamez Gonzalez et al. 2024).

Here, we investigated the effect of chemical synchronization on the lifespan of C. elegans at different temperatures. We conducted parallel lifespan assays on wild-type worms collected by egg-laying and chemical synchronization at 16, 20, and 25°C. In our multiple independent assays, we couldn’t observe any chemical synchronization condition that resulted in a lifespan significantly different from that of laid-egg animals at every tested temperature. Moreover, there was no difference among the six chemical synchronization conditions (Figure 7A, E, and I). From the pooled analysis, no effect was observed from chemical synchronization at 16°C (Figure 7B–D), 20°C (Figure 7F–H), and 25°C (Figure 7J–L). These results strongly support the conclusion that chemical synchronization does not affect the lifespan of wild-type N2 at widely tested temperatures.

Figure 7.

Chemical synchronization does not affect the lifespan of wild-type N2 at 16, 20, and 25 °C. Survival curves of wild-type N2 at 16 °C (A-D), 20 °C (E-H), or 25 °C (I-L). Animals were egg-laid or chemically synchronized using weak (W), moderate (M), or strong (S) bleaching solutions for 5 s (5s) or 5 min (5 m). Raw survival data (A, E, and I) were pooled depending on treatment durations (B, F, and J), bleaching solution concentrations (C, G, and K), and chemical synchronization (CS) regardless of duration or solution (D, H, and L), as described in the ‘Statistics’ section in the Materials and Methods. (A-D) n = 308–572 worms throughout N = 3–4 independent assays, (E-H) n = 181–473 with N = 3–5, and (I-L) n = 407–837 with N = 4–5. (A-L) ns > 0.01 for all comparisons from Log-rank tests with Bonferroni correction.

The lifespan of short-lived daf-16 (mu86) does not change with chemical synchronization at 20 and 25°C, but at 16°C

Next, we asked whether a short-lived mutant that has never been tested is affected by chemical synchronization. We selected daf-16(mu86), a loss-of-function mutant in the C. elegans homolog of the forkhead box transcription factors class O (FoxO) daf-16, for the test (Zecic and Braeckman 2020). First, a lifespan assay with synchronized daf-16(mu86) was performed at 20 and 25°C, the temperatures commonly used to monitor the shortened lifespan. The lifespan of daf-16(mu86) from six chemical synchronization conditions did not differ from that of daf-16(mu86) from egg-laying at 20 and 25°C (Supplementary Data S4A and S4E). Although not statistically significant, it is worth noting that the daf-16(mu86) lifespan differences between some synchronization conditions at 20°C (egg-laid vs ‘Weak, 5 s’, ‘Strong, 5 s’ vs ‘Weak, 5 s’; Supplementary Data S4A) and 25°C (‘Weak, 5 s’ vs ‘Moderate, 5 min’, ‘Moderate, 5 s’ vs ‘Moderate, 5 min’; Supplementary Data S4E) are more noticeable than the N2 lifespan differences (Figure 7E and I). The pooled analysis revealed no effect of chemical synchronization on the lifespan of daf-16(mu86) at 20°C (Supplementary Data S4B-S4D) and 25°C (Supplementary Data S4F-S4H). These results indicate that chemical synchronization may enhance lifespan variation in short-lived daf-16(mu86) at relatively low temperatures, at which worms usually live longer.

In C. elegans, lifespan extension at low temperatures involves activation of the cold-sensitive TRP channel, TRPA-1, and DAF-16 plays a critical role in TRPA-1-mediated lifespan extension at low temperatures, such as 15°C (Xiao et al. 2013). Therefore, we measured the lifespan of daf-16(mu86) at 16°C. We initially noticed that the lifespan of egg-laid daf-16(mu86) was not significantly different from that of those collected from chemical synchronization (Figure 8A). However, some chemical synchronization conditions yielded significantly different lifespans from other conditions. Specifically, ‘Weak, 5 s’ and ‘Moderate, 5 s’ chemical synchronization produced daf-16(mu86) that lived significantly longer than both ‘Strong, 5 s’ and ‘Strong, 5 min’ chemical synchronization (Figure 8A). The pooled data analysis revealed that the concentration of the bleaching solution may be a significant factor contributing to the differences in the chemical synchronization. The daf-16(mu86) synchronized with ‘Weak’ or ‘Moderate’ concentrations lived significantly longer at 16°C than those synchronized with ‘Strong’ concentrations (Figure 8C). The results suggest that chemical synchronization may increase variation in the lifespan of the short-lived daf-16 mutant, which plays a critical role in sensing cold.

Figure 8.

Chemical synchronization may affect the longevity of daf-16(mu86) at 16°C. Survival curves of daf-16(mu86) at 16 °C. Animals were egg-laid or chemically synchronized using weak (W), moderate (M), or strong (S) bleaching solutions for 5 s (5s) or 5 min (5 m). The raw survival data (A) were pooled by treatment duration (B), bleaching solution (C), and chemical synchronization (CS), regardless of duration or concentration (D). See the ‘Statistics’ section in the Materials and Methods for further details. (A–D) n = 183–304 worms throughout N = 3 independent assays. (A–D) * < 0.01, ** < 0.005, *** < 0.001, **** < 0.0001. Log-rank tests with Bonferroni correction.

Discussion

Chemical synchronization using a household bleach solution is a widely accepted method for preparing large quantities of synchronized C. elegans. This method takes advantage of the extreme durability of the C. elegans eggshell, a multilayered structure that protects the embryo from harsh environmental conditions. A C. elegans embryo is immediately surrounded by an inner vitelline membrane that must be intact to ensure proper development, while the outer chitinous layer is dispensable for successful development to yield a normal animal (Schierenberg and Junkersdorf 1992). The chitinous layer is resistant to mechanical force and chemicals, allowing it to bleach and break down the gravid adults and release the fertilized eggs, which mostly develop normally and hatch. It was widely accepted that worms from bleached eggs are indistinguishable from those from laid eggs. However, it has been questioned whether experiments involving bleaching can be recapitulated as those using laid eggs. In this study, we tested a hypothesis that chemical synchronization using bleach may influence the outcomes of experiments investigating developmental and behavioral phenotypes of C. elegans. Our results showed that chemical synchronization can affect hatch rate and developmental rate, brood size, and morphology in C. elegans. However, no difference was found in lifespan and sensory behaviors mediated by amphid neurons.

Previous studies have shown that chemical synchronization reduces the mechanical rigidity (Krenger et al. 2020) and egg viability (Baris Atakan et al. 2020; Verdu et al. 2022). We also observed that chemical synchronization reduced hatch rate (Figure 1B and C), delayed development (Figure 2), changed morphological characteristics (Figure 3), decreased brood size (Figure 5), and led to a difference in the daf-16(mu86) lifespan at 16°C between different conditions (Figure 8). It is interesting, however, that chemical synchronization did not affect the developmental potential between early- and later-stage eggs (Figure 4), the pharyngeal pumping rate (Supplementary Data S1), chemotactic behavior toward benzaldehyde, isoamyl alcohol, 2-butanone (Figure 6), diacetyl (Supplementary Data S2), copper ion, and glycerol-induced hyperosmolarity (Supplementary Data S3), the lifespan of N2 wild-type at 16, 20, and 25°C (Figure 7), and that of daf-16(mu86) at 20 and 25°C (Supplementary Data S4).

The gravid C. elegans uterus is filled with eggs containing embryos at various stages, from recently fertilized to more developed. Therefore, the physiological age of the collected eggs after chemical synchronization varies widely. To highly synchronize the population, researchers often let larvae from bleached eggs starve to get arrested at the L1 stage, at which young worms enter a diapause state where gene expression shifts and stops post-embryonic development (Baugh 2013). However, this experimental tactic may significantly change worms’ physiology. Acute starvation for 6 days changes the small RNA expression patterns in the starved P0 and F3 generations and increases the lifespan of the F3 animals (Rechavi et al. 2014). Moreover, 5 h of starvation at the L1 stage delays adult molting, reduces brood size, and changes the number of offspring produced daily (Perez et al. 2017). To clearly investigate the bleaching effect on the worms’ development and behavior, we isolated the L4-stage animals after hatching without starvation. The L4 preparation also excluded immature animals produced from late-hatching eggs or slow-developing animals (Figure 2).

Another factor that must be carefully considered in chemical synchronization is the condition of gravid adults treated with the bleaching solution. The age of hermaphrodites affects the characteristics of their offspring. The offspring of day 1 adult-stage animals exhibit slower developmental speed, smaller body size and brood size than those of day 2 and 3 adult-stage animals (Perez et al. 2017). The progeny of day 5 adult-stage animals are bigger than those of day 2 adult-stage animals (Zhang et al. 2023). Various genes involved in cuticle formation determine and influence body plans and structure, and mutant worms with their cuticle structure altered can be sensitive to bleaching solutions (Gravato-Nobre et al. 2005; Wang et al. 2024). Therefore, indiscriminate use of animals with varying starvation histories, ages, or strains in chemical synchronization may result in a population of animals inconsistently exhibiting traits. To avoid these unwanted consequences, we strictly used plates containing day 2 adult-stage animals that were well-fed for generations.

As the assembly of eggshell is completed by anaphase II (Olson et al. 2012) and the eggshell after the process is known not to be modified further, it may have been reasonable to assume that the eggshell structures of early – and later-stage embryos are not different. If the chitin layer of the eggshell perfectly protects the fertilized eggs, the hatch rate of early- and later-stage embryos must have been no different. However, in this study, even a 5-second treatment with the weak bleaching solution significantly lowered the hatch rate of the early-stage eggs (Figure 1B). We also observed that the hatch rate of early-stage embryos directly isolated from hermaphrodites was 95.3%. These findings suggest that embryos at earlier stages of development should be more susceptible to environmental stress, such as chemical exposure, than those at later stages. It was also reported that 2- and 3-fold embryos showed relatively higher heat tolerance than pre-2-fold embryos (Jhaveri et al. 2025). The reduced hatch rate after chemical synchronization was also observed in other studies (Baris Atakan et al. 2020; Verdu et al. 2022). As chemical synchronization can damage the chitin layer and impair the mechanical rigidity of the C. elegans egg (Krenger et al. 2020), it is recommended not to use chemical synchronization in experiments using mutants displaying eggshell defects or drug treatments that can increase the permeability of the eggshell.

While carrying out this study, we occasionally observed several abnormal worms collected from chemical synchronization. Under all chemical synchronization conditions, some eggs hatched much later than others, even as much as a couple of days, but delayed hatching was never observed in egg-laying synchronization. The embryos from the chemical synchronization process may enter a state of suspended animation, temporarily ceasing development under unfavorable environmental conditions, such as a low supply of oxygen caused by bleach treatment, which may make the eggshell less permeable (Padilla et al. 2002; Padilla and Ladage 2012). The embryos at earlier stages of development may be more susceptible to stress and damage, as their hatch rate was significantly lower than that of later-stage embryos (Figure 1B and C). Another abnormal case is a post-embryonic N2 wild-type animal with a mild roller phenotype. These rare worms were observed only during the ‘Strong, 5 min’ chemical synchronization, and these animals moved slowly with frequent rolling. The roller phenotype results from malformed cuticles and hypodermis caused by mutations in cuticle collagens, such as rol-6 (Kramer and Johnson 1993). The presence of a roller worm suggests that chemical synchronization may result in a change in gene expression pattern that can affect the worms’ cuticle structure.

Chemical synchronization with different conditions affected the morphological characteristics of wild-type worms differently (Figure 3A, C, E, and G). However, it did not yield clear differences across the tested characteristics in concentrationor treatment-duration-dependent pooled data sets. The differences decreased in the pooled assay (Figure 3B, D, F, and H). This kind of variability between multiple chemical synchronization experiments was also observed in the viability of chemically synchronized eggs (Baris Atakan et al. 2020). These suggest that chemical synchronization tends to lead to variable levels of change or trend among the collected animals.

In this study, we had to repeat chemotaxis assays to conclude statistical insignificance. Chemotaxis assays of chemically synchronized animals using benzaldehyde (Figure 6A), 2-butanone (Figure 6E), and diacetyl (Supplementary Data S2A) required 3–6, 3–5, and 3–5 replicates to achieve clear resolution. In contrast, for the chemotaxis assay using isoamyl alcohol, the number of replicates ranged from 7 to 16 to reach statistical insignificance with confidence (Figure 6C). Therefore, we suggest that the option of a synchronization method should be carefully considered when using certain volatile chemicals, especially isoamyl alcohol, in chemotaxis assays. On the other hand, the droplet assay using copper ions and glycerol-induced hyperosmolarity required only 3–4 replicates (Supplementary Data S3). This implies that the nociceptive response of C. elegans is so robust that it produces a clearer behavioral response than other behaviors induced by volatile chemicals.

Chemical synchronization does not affect the longevity of wild-type animals at any of the tested temperatures (16, 20, and 25°C; Figure 7). Apart from many genes that regulate lifespan (Uno and Nishida 2016), it has become clear that cold temperature (15°C) and warm temperature (25°C) induce lifespan shifts through distinct pathways. For example, a nuclear activity of DAF-16 mediated by the TRP channel (Xiao et al. 2013) and fatty acid modification mediated by MDT-15 (Lee et al. 2019) are required for enhanced longevity at cold temperatures, while neuron-mediated signaling regulates lifespan at warm temperatures (Vakkayil and Hoppe 2022). Therefore, tightly regulated interactions of multiple pathways controlling the lifespan of C. elegans must be responsible for the unchanged longevity of chemically synchronized N2 animals. On the other hand, the short-lived daf-16(mu86) mutant animals prepared with chemical synchronization show much variation in lifespan at cold temperatures, but not at relatively warm temperatures (Figure 8 and Supplementary Data S4). DAF-16 is required for longevity at cold temperatures (Xiao et al. 2013). Therefore, it is recommended to test synchronization methods in parallel for the lifespan assay with mutant animals, particularly those of genes involved in the temperature-sensitive process.

This study aims to provide a better understanding of the chemical synchronization of C. elegans and its effects in common experiments. Chemical synchronization has been widely used in various fields of study using C. elegans. However, the harsh treatment has been applied using a variety of modified protocols, and its effects have not been thoroughly investigated. Performing parallel experiments under six chemical synchronization conditions and with egg-laying as a control, we examined the effects of chemical synchronization on several embryonic and post-developmental traits. We found that chemical synchronization can affect experimental results and yield high variation; thus, careful consideration is required. To avoid incorrect results and unwanted variation in experiments using chemical synchronization, it may be necessary to repeat experiments sufficiently and use a standardized protocol tested by the experimenters.

Funding Statement

This study was supported by the National Research Foundation of Korea (NRF) grant (2018R1A2A3074987 to S-K. L.; RS-2024-00335112 to H.S. K). Strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Declaration of generative AI use

The authors report that generative AI was not used in their research or preparation of this manuscript.

Supplemental Material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19768354.2026.2719247.