Brief guide to assays for measuring health parameters using Caenorhabditis elegans

Abstract

Assessing health parameters is essential for understanding the decline of physiological functions during aging. The nematode Caenorhabditis elegans serves as a powerful model in aging research. This paper introduces representative methods for evaluating health parameters using C. elegans and provides quantitative indicators of functional declines during aging. This manuscript will be useful for researchers unfamiliar with healthspan studies using C. elegans and may help perform physiological experiments for healthy aging.

INTRODUCTION

Aging is accompanied by a progressive decline in physiological functions, leading to increased susceptibility to diseases and reduced overall fitness (Hwang and Song, 2023, Lopez-Otin et al., 2023, Ma et al., 2023, Mun et al., 2024, Park et al., 2024, Son et al., 2019, Yan et al., 2024). The nematode Caenorhabditis elegans is an invaluable model organism for research on animal physiology, including aging, because of its short life cycle, convenience for maintenance, and high genetic homology with humans (Ji et al., 2024, Kim et al., 2024, Kwon et al., 2023, Lee et al., 2024). For example, C. elegans lifespan and survival assays have been performed for aging and stress response research (Gaglia et al., 2012, Jung et al., 2020, Kim et al., 2023, Park et al., 2021, Park et al., 2017, Seo et al., 2015). C. elegans further serves as a powerful system for evaluating healthspan (Park et al., 2021). While human healthspan is defined as the period free from chronic diseases and major functional impairments, C. elegans healthspan measurement relies on age-dependent declines in physiological performance as a surrogate. Nevertheless, C. elegans health parameters offer valuable insights into aging mechanisms relevant to human health, because of many evolutionarily conserved signaling pathways that influence aging and longevity (Lopez-Otin et al., 2023, Son et al., 2019). Measurement of C. elegans healthspan requires defining various health parameters, which reflect age-dependent declines in physiological processes, such as locomotion, reproductive capacity, and stress resistance (Keith et al., 2014, Kirchweger et al., 2023, Son et al., 2019).

Here, we briefly describe key methodologies for measuring physiological health status in C. elegans. We introduce assays covering neuromuscular function assessment through crawling and swimming (thrashing), feeding evaluation via pharyngeal pumping, and defecation cycle observation (Fig. 1). In addition, we cover assays for structural integrity through intestinal permeability analysis, stress resistance under abiotic and biotic conditions, and reproductive health measurement based on brood size. By providing a concise guide to these assays, this manuscript functions as a resource for researchers investigating age-dependent functional changes in C. elegans, one of the most powerful model systems for studying aging and healthy longevity. While this MiniResource offers a concise overview of representative assays, further details are available in other review articles (Hart, 2006, Keith et al., 2014, Park et al., 2017, Raizen et al., 2012, Salazar et al., 2023, Son et al., 2019, Wibisono and Sun, 2022).

Fig. 1.

A schematic of representative assays for measuring health parameters using Caenorhabditis elegans. Crawling and swimming (thrashing) assays assess the S-shape bending phenotypes and C-shape bending frequency of C. elegans, respectively. Feeding (pharyngeal pumping) assays measure the feeding cycle as an indicator of food intake efficiency. Defecation assays determine defecation cycle length or frequency to evaluate digestive activity. Intestinal integrity is examined using blue dye leakage with intestinal permeability assays, which are known as the Smurf assay. Stress resistance is assessed under abiotic and biotic conditions, measuring responses to environmental stressors and pathogenic infections, respectively. Reproductive capacity is quantified through brood size measurement by counting hatched progeny.

Crawling and Swimming (Thrashing) Assays

Crawling assay on solid media and swimming (thrashing) assay in liquid media are two representative motility assays used for C. elegans (Hart, 2006, Parida, 2022). Crawling assay is convenient because of no requirement for additional preparations when the animals are cultured on solid media, but is relatively difficult in analyzing movement dynamics compared to the swimming assay (Korta et al., 2007). For the swimming assay, researchers obtain a consistent C-shape bending movement of C. elegans in liquid media for a certain period, usually 30 seconds to 1 minute (Hart, 2006, Parida, 2022). By quantifying the decline of bending frequency on solid and in liquid during aging, crawling and swimming assays can be used for evaluating the healthspan of C. elegans (Park et al., 2021, Son et al., 2019).

Crawling and swimming assays are performed using manual or automated methods (Hart, 2006, Kirchweger et al., 2023, Parida, 2022). For manual crawling assay, C. elegans at specific ages are transferred onto plates without buffer, ensuring that the animals are exposed to minimal external variables. The animal behaviors are then recorded and analyzed for S-shape bending and locomotion phenotypes. Similarly, with the manual approach of swimming assays, researchers transfer C. elegans at specific ages onto the plates containing buffer, and wait for equilibration, followed by recording of animal movements to measure C-shape bending frequency. Automated crawling and swimming analysis using computer vision software are convenient tools for tracking the movements of C. elegans and are suitable for high-throughput studies (Lavorato et al., 2021).

Feeding (Pharyngeal Pumping) Assays

Assays that measure feeding (pharyngeal pumping) rates quantify healthspan by assessing age-related declines in food uptake rates, caused by bacterial plugging or reduced pharyngeal function (Croll et al., 1977, Park et al., 2021, Raizen et al., 2012). For the feeding rate assay, researchers count the contractions of the pharynx of C. elegans at specific ages under a stereomicroscope. In addition, because the animal under analysis may constantly move, it is crucial to track the movement of C. elegans grinder in the pharynx under a stereomicroscope, often by recording and then analyzing the videos for accurate quantification (Raizen et al., 2012, Shanmugam and Kapahi, 2024). Automated feeding rate analysis overcomes laborious work required for increasing replicates, suitable for large-scale experiments with consistency and reproducibility (Rodriguez-Palero et al., 2018, Bonnard et al., 2022).

Defecation Assays

The defecation process occurs through posterior body muscle contraction, anterior body muscle contraction, and is completed by expulsion muscle contraction (Hart, 2006). The defecation cycle is defined as the time between 2 intestinal expulsions. Young adult wild-type C. elegans usually exhibit a regular defecation cycle with intervals of approximately 40 seconds and a standard deviation of 2 to 3 seconds. However, defecation intervals increase during aging with multiphase (Bolanowski et al., 1981), indicating that defecation cycle length can be used for analyzing healthspan. Synchronized animals at a specific age are transferred to fresh plates and adapted for a proper period (Wibisono and Sun, 2022). Researchers then measure the defecation cycle length or count the number of defecation cycles periodically for a certain period, until the expulsions stop being visible. Subsequently, the defecation rate is calculated over the observed period.

Intestine Permeability Assays

The intestine of C. elegans forms junctional complexes to maintain a condensed structure, thereby functioning as a barrier against harmful substances such as pathogenic bacteria (Markovich et al., 2024). However, aging weakens the intestinal barrier integrity in C. elegans (Salazar et al., 2023). Thus, monitoring intestinal leakage by using specific substances such as blue dyes can be used as a parameter to measure healthspan, which is often referred to as the Smurf assay (Gelino et al., 2016). To evaluate intestinal barrier integrity, researchers collect C. elegans at specific ages and incubate the animals for several hours in a liquid culture containing Escherichia coli OP50 (laboratory food) mixed with a blue dye. Residual dye and bacteria are then removed through multiple washes, followed by mounting the animals or transferring the animals to fresh plates to measure the blue dye in the body cavity. The leakage of blue dye from the intestinal lumen into the body cavity serves as an indicator of intestinal integrity. Irinotecan, which impairs intestinal integrity, is a useful positive control for evaluating proper dye leakage patterns in the animals (Zhu et al., 2019).

Abiotic and Biotic Stress Assays

Aging leads to a gradual decline in resistance to abiotic and biotic stressors, indicating that measuring stress resistance at specific ages can be used for evaluating health status in C. elegans (Keith et al., 2014, Park et al., 2017). Abiotic stress assays assess physiological responses of the animals to environmental stressors, including thermal stress, oxidative stress, osmotic stress, heavy metal stress, ER stress, and UV radiation stress (Cho et al., 2024, Choi, 2023, Gao et al., 2024, Jaishankar et al., 2014, Jung et al., 2023, Keith et al., 2014, Lee et al., 2015, Lee et al., 2023, Lithgow et al., 1994, Murakami and Johnson, 1996, Murray et al., 2007, O'Neil and Rose, 2006, Park et al., 2023, Rodriguez et al., 2013, Sies et al., 2017, Song et al., 2024; Vilchez et al., 2014). For measuring each stress resistance, researchers transfer C. elegans at specific ages onto the plates with stressors and then evaluate the survival rate of the animals, ensuring the control of environmental conditions to maintain the accuracy and consistency of the results (Keith et al., 2014, Park et al., 2017). Biotic stress assays are conducted by transferring animals at specific ages onto plates with pathogenic bacteria or fungi. Researchers then score the survival of the animals at specific ages periodically and calculate the survival rate (Han et al., 2024, Han et al., 2016, Yang et al., 2011). Further details of stress assays using C. elegans are described in other reports (Park et al., 2017, Kwon and Lee, 2025).

Brood Size Measurement Assays

Measurement of brood size, quantification of the total number of hatched live progeny, evaluates reproductive capacity as a health parameter in C. elegans (Kirchweger et al., 2023, Son et al., 2019). Brood size measurement allows simultaneous assessment of the total progeny number, reproductive span, and the proportion of progeny produced per day. Researchers isolate each L4 stage animal (parental; P₀) onto a fresh plate and grow the animal overnight for allowing to lay eggs. Each P₀ animal is then transferred onto a fresh plate periodically until the animal stops laying eggs (Castro Torres et al., 2022, Hart, 2006). The hatched (filial; F₁) progeny that reach the L4 or young adult stage on the plates are counted. Researchers retain the plates for an additional 1 or 2 days because of the possibility of delayed hatching or development.

CONCLUDING REMARKS

In this paper, we introduce representative assays for measuring health parameters in C. elegans, providing a brief and practical resource for researchers who are unfamiliar with healthspan research. Depending on experimental purposes, detailed protocol modifications are required. Integrating cellular and molecular biomarkers such as lipofuscin accumulation (Son et al., 2019) and protein homeostasis markers (e.g., hsp-16.2::GFP [Rea et al., 2005]) can further improve the evaluation of healthspan by helping bridge the gap between organismal healthspan and cellular aging processes. In addition, as previous studies have reported cases where increased healthspan does not necessarily correlate with extended lifespan, it is recommended to assess lifespan with healthspan to obtain a comprehensive understanding of aging procedures (Bansal et al., 2015).

Funding and Support

This work was supported by the National Research Foundation of Korea grant funded by the Korea government (MSIT) (RS-2024-00408712) to S.J.V.L.

Author Contributions

S. H., J. L., and S.J.V.L. wrote the manuscript.

CRediT authorship contribution statement

Seung-Jae V. Lee: Writing – review & editing, Supervision. Seungjae Hwang: Writing – review & editing, Writing – original draft. Jongsun Lee: Writing – review & editing.

Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work the authors used ChatGPT 4o (OpenAI) in order to improve language clarity of the manuscript. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Declaration of Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.