Caenorhabditis elegans as an emerging model in food and nutrition research: importance of standardizing base diet

Abstract

As a model organism that has helped revolutionize life sciences, Caenorhabditis elegans has been increasingly used in nutrition research. Here we explore the tradeoffs between pros and cons of its use as a dietary model based primarily on literature review from the past decade. We first provide an overview of its experimental strengths as an animal model, focusing on lifespan and healthspan, behavioral and physiological phenotypes, and conservation of key nutritional pathways. We then summarize recent advances of its use in nutritional studies, e.g. food preference and feeding behavior, sugar status and metabolic reprogramming, lifetime and transgenerational nutrition tracking, and diet–microbiota–host interactions, highlighting cutting-edge technologies originated from or developed in C. elegans. We further review current challenges of using C. elegans as a nutritional model, followed by in-depth discussions on potential solutions. In particular, growth scales and throughputs, food uptake mode, and axenic culture of C. elegans are appraised in the context of food research. We also provide perspectives for future development of chemically defined nematode food (“NemaFood”) for C. elegans, which is now widely accepted as a versatile and affordable in vivo model and has begun to show transformative potential to pioneer nutrition science.

Keywords:

• Nematode food
• axenic culture
• nutritional memory
• microbiota
• food preference
• pharyngeal pumping
• healthspan
• transgenerational tracking

Introduction

Humans and animals require sufficient nutrients to support their life activities, including growth, development, reproduction, movement and survival. A balanced diet ensures the intake of essential nutrients that provide adequate energy and substances for these activities, while either deficiency or excess of nutrients may cause suboptimal functioning of the body, leading to metabolic stress and associated disorders (GBD 2017 Diet Collaborators 2019; Lepre et al. 2021). In the last few decades, lifestyle routines and eating habits have drastically changed, including substantial shift from home‐made to industrial food, due to the rapid development of food production and the globalization of food supply. This transition is accompanied by significant changes in dietary intake with a modified nutritional pattern thanks to the increased availability of food, especially energy-dense food. The resulting nutritional imbalance and energy surplus, as opposed to evolutionarily adapted mechanisms of food shortage, are particularly alarming as early suboptimal nutrition may have long-term health consequences several decades even generations later (Gentile and Weir 2018; Jiménez-Chillarón et al. 2012; Robinson 2018). Indeed, increasing evidence suggests that nutritional memory from earlier life stages of an individual per se, as well as transgenerational metabolic memory of ancestral food experience, may have important impacts on metabolic physiology, including food consumption and nutritional balancing strategies, which in turn will affect health and disease (Barrès and Zierath 2016; Fontana and Partridge 2015; Hahn et al. 2019; Perez and Lehner 2019). However, convenient models for nutrition studies in such contexts still need further development to unravel long-term and cross-generational beneficial as well as adverse effects and underlying molecular mechanisms, including signaling, transcriptional and epigenetic regulations.

The experimentally tractable nematode Caenorhabditis elegans has been widely used as a model animal that has helped revolutionize our understanding in virtually all aspects of life sciences for half a century (C. elegans Sequencing Consortium 1998), and has also emerged as a favorable model in food and nutrition studies (Cypser, Kitzenberg, and Park 2013; Gottschling and Döring 2019; Shen et al. 2018; Zečić, Dhondt, and Braeckman 2019). As summarized in Table 1, C. elegans has been used in recent years to investigate almost every category of nutrients, including carbohydrates, proteins, lipids, vitamins and minerals. For example, wild-type as well as various transgenic C. elegans models are used to assess the bioactivities of polysaccharides, peptides and other extracts derived from food (e.g. Chen et al. 2020; Guo et al. 2020; Jia et al. 2018; Li et al. 2011; Lu et al. 2021; Wang et al. 2016; H. Zhang et al. 2012; J. Zhang et al. 2016; Zhong et al. 2021). Given the data available thus far, it is entirely plausible to foresee the transformative power of C. elegans in pioneering nutrition science, e.g. lifelong monitoring and transgenerational tracking of nutritional status. To appreciate the groundbreaking potential, however, a number of inadvertent compromises must be fully realized to forestall misuses of C. elegans models. Here we focus on the feasibility and advantages as well as limitations and solutions of using C. elegans as a “dietary” model in nutritional studies.

Table 1. Bioactivities of food nutrients and functional components investigated in C. elegans models.^a

Nutrient/food component	Functionality/dysfunctionality and underlying mechanisms
Carbohydrates
Glucose	Lifespan and healthspan (Gusarov et al. 2017; Jung et al. 2020; Lee, Murphy, and Kenyon 2009; Liggett et al. 2015; Riedinger et al. 2018; Schlotterer et al. 2009; Schulz et al. 2007; Seo et al. 2018); stress resistance (Gusarov et al. 2017; Tauffenberger and Parker 2014); reproduction (Teshiba, Miyahara, and Takeya 2016); immunity (Li et al. 2018); neuronal dysfunction (Salim and Rajini 2017); mitochondria (Alcántar-Fernández et al. 2019; Schulz et al. 2007); autophagy (Franco-Juárez et al. 2018); lipid biosynthesis and fat storage (Garcia et al. 2015; Zheng et al. 2014); glycogen storage (Gusarov et al. 2017; Seo et al. 2018); body increase (Alcántar-Fernández et al. 2018)
Fructose	Lifespan and healthspan (Lodha et al. 2022; Zheng et al. 2017); fat storage (Zheng et al. 2014); body increase (Ke et al. 2021)
Sucrose	Lifespan and healthspan (Wang, Zhang, et al. 2020); reproduction, growth and development (Wang, Zhang, et al. 2020); fat storage (Zheng et al. 2014, 2017); body increase during the spawning period (Wang, Zhang, et al. 2020)
Oligosaccharides	Lifespan and healthspan (Fang et al. 2019; Wu et al. 2020; Zhong et al. 2021); oxidative stress (Fang et al. 2019; Wu et al. 2020; Zhong et al. 2021); hypoglycemia (Wu et al. 2020); insulin resistance (Wu et al. 2020); locomotion (Fang et al. 2019); neuroprotection (Zhong et al. 2021)
Polysaccharides	Lifespan and healthspan (Li et al. 2016; Wang et al. 2015; Zhang et al. 2012; Zhong et al. 2021); oxidative stress (Li et al. 2011, 2016; Xiang et al. 2017; Zhang et al. 2016; Zhong et al. 2021); mitochondria (Zhang et al. 2016); fat storage (Bai et al. 2021; Lin et al. 2020); food-sensing and uptake behavior (Li et al. 2016; Lin et al. 2020); neuroprotection (Li et al. 2016; Wang, Yi, and Zhao 2018; Xiang et al. 2017; H. Zhang et al. 2012; J. Zhang et al. 2016; Zhong et al. 2021)
Amino acids and proteins
Free amino acids	Lifespan and healthspan (Edwards et al. 2015; Liu et al. 2019); stress resistance (Edwards et al. 2015); chemotaxis (Bian et al. 2018); sensory neurons (Metzler et al. 2013); absorption of nutrient amino acids (Metzler et al. 2013); amino acid deficiency (Martin et al. 2011)
Peptides and proteins	Lifespan and healthspan (Chaubey et al. 2020; Chen et al. 2020; Guo et al. 2020; Wang et al. 2016); oxidative stress (Chen et al. 2020; Guo et al. 2020; Jia et al. 2018; Lu et al. 2021; Wang et al. 2016); neuroprotection (Chaubey et al. 2020)
Lipids and fatty acids
Oleic, arachidonic, eicosapentaenoic and docosahexaenoic acids	Oxidative stress and lifespan (Han et al. 2017; Lynn et al. 2015; O’Rourke et al. 2013; Rangaraju et al. 2016); reproduction (Lynn et al. 2015; Shemesh et al. 2017); nutrient balance (Lynn et al. 2015); alcohol response (Raabe et al. 2014); neuroprotection (Shashikumar et al. 2015); movement (Shashikumar et al. 2015)
Linoleic and linolenic acids	Lifespan (S. Wang, Xue, et al. 2020); oxidative stress resistance (S. Wang, Xue, et al. 2020); fat level (Colmenares et al. 2016); reproduction (Deline et al. 2015; Shemesh et al. 2017)
Stearic acid	Lifespan, reproduction, growth and development (X. Wang, Xue, et al. 2020)
Fatty acid extracts	Lifespan, oxidative stress resistance (S. Wang, Xue, et al. 2020)
Vitamins
B12	Metabolic plasticity and regulation (Giese et al. 2020; Watson et al. 2016); mitochondrial function (Revtovich, Lee, and Kirienko 2019; Wei and Ruvkun 2020); reproduction (Watson et al. 2014); development (Na et al. 2018; Watson et al. 2014); oxidative stress (Bito et al. 2017); memory retention (Bito et al. 2017); body increase in axenic medium (Surafel et al. 2018)
C	Lifespan (Pallauf et al. 2013; Polyak et al. 2018); oxidative stress (Gourgou and Chronis 2016; Polyak et al. 2018)
D	Lifespan (Mark et al. 2016; Rangaraju et al. 2016); oxidative stress (Mark et al. 2016); Aβ-induced toxicity and paralysis (Leiteritz et al. 2021; Mark et al. 2016)
E	Lifespan (Polyak et al. 2018; Sakamoto et al. 2020; Schlotterer et al. 2021); oxidative stress (Polyak et al. 2018; Sakamoto et al. 2020); neuroprotection (Schlotterer et al. 2021)
Folate	Lifespan (Virk et al. 2016; Rathor et al. 2015); oxidative stress (Koseki et al. 2020; Rathor et al. 2015); body increase (Virk et al. 2016)
Minerals
Zinc	Lifespan and healthspan (Kumar et al. 2016); reproduction (Mendoza et al. 2017); development (Kumar et al. 2016); lipid metabolism (Zhang, Holdorf, et al. 2017)
Iron	Lifespan and healthspan (Jenkins et al. 2020; Schiavi et al. 2015); oxidative stress (Valentini et al. 2012; J. Zhang, Li, et al. 2019); mitochondrial stress (Schiavi et al. 2015); development (Romney et al. 2011; J. Zhang, Li, et al. 2019); immunity (Rajan et al. 2019)
Calcium	Mitochondrial and oxidative stress (Momma et al. 2017; Sarasija and Norman 2015; Young et al. 2019); neuronal functions (Liu et al. 2018; Offenburger, Jongsma, and Gartner 2018; Young et al. 2019)
Selenium	Lifespan and healthspan (Köhnlein et al. 2020); oxidative stress (Köhnlein et al. 2020; Rohn et al. 2019); neurodegeneration (Estevez et al. 2014); immune response (Li et al. 2014)

^aExamples selected from studies published in the last decade.

Experimental strengths of C. elegans as a nutritional model

As a major model organism in life sciences and biomedical research, C. elegans is powerful at both molecular and organismal levels and has a number of advantages such as small size (∼1 mm in length with 959 cells for adult hermaphrodites), rapid life cycle (∼3 days), short lifespan (∼3 weeks), convenience of laboratory maintenance (agar plates or liquid cultures), ease of experimental manipulation (e.g. 96- and 384-well plates), and genetic similarity to human beings (containing potential counterparts of most human disease genes and signaling networks). Despite its small size, this seemingly simple nematode model does have diverse tissues, e.g. nervous, muscle, intestinal, and reproductive systems. In addition, access to vast online resources is publicly available, including specialized knowledge as well as genetic and strain resources such as WormBook (http://www.wormbook.org), WormBase (https://wormbase.org) and CGC (https://cbs.umn.edu/cgc/home; i.e. Caenorhabditis Genetics Center).

Take as an example healthspan, which is becoming a central topic in aging and nutritional studies (Lee et al. 2021; Olshansky 2018; Scott, Ellison, and Sinclair 2021; Seals, Justice, and LaRocca 2016; Wickramasinghe et al. 2020). Healthspan can be defined as the period of life that an organism is free from major diseases and disabilities. As a leading model in aging research, C. elegans has been increasingly used to help distinguish between lifespan and healthspan (e.g. Bansal et al. 2015; Huang, Ma, et al. 2022; Möller et al. 2020), both of which are essential aspects of aging that can be regulated by dietary manipulation (Lee et al. 2021; Wickramasinghe et al. 2020). In consensus with the concept in other organisms, three main categories of health indicators have been developed in C. elegans healthspan, i.e. cell and tissue integrity, tissue and organ function, and physiological and stress resilience (Bansal et al. 2015; Hahm et al. 2015; Keith et al. 2014; Koopman et al. 2020).

A number of behavioral and physiological phenotypes have been adopted for healthspan studies in C. elegans, including locomotion, body bends, pharyngeal pumping, and body size (Bansal et al. 2015; Ewald, Castillo-Quan, and Blackwell 2018; Seo et al. 2018). The highly coordinated and tightly regulated pharyngeal pumping is an essential action of C. elegans for food intake. Illustrated in Figure 1A is a representative image of C. elegans pharynx. Feeding is accomplished by cycles of contraction and relaxation in the pharynx, where the corpus sucks in and concentrates food while the terminal bulb grinds it up and transports it to the intestine (Avery and Shtonda 2003; Avery and You 2012). The pharyngeal pumping rate, which is modulated by food availability and thus a reliable measure of nutritional status, can be measured by directly counting the visible pumps under a microscope and used as a reliable and quantitative indicator of C. elegans health (Lemieux et al. 2015; Williams et al. 2019). Another essential indicator of health is body size and body shape, which is of utmost importance for food and nutrition studies. We have previously shown that paeoniflorin is able to delay progressive paralysis of C. elegans caused by the expression of amyloid-β peptide (Aβ), but it does not affect their body size as analyzed by a COPAS Biosort system using large number of animals, including both time of flight (TOF; equivalent to nematode length) and extinction (EXT; indicative of internal structure of nematodes) (Figure 1B), implying no compromise on the health of C. elegans (Ai et al. 2018).

Figure 1. Pharyngeal pump and body size of Caenorhabditis elegans. (A) Microscopy of pharynx indicating pharyngeal pumping and food transport in C. elegans. A representative image of pharynx (about 100 × 20 μm in adult hermaphrodites) is shown to indicate the neuromuscular pump that connects to the mouth at the anterior end via the corpus and to the intestine at the posterior end via the terminal bulb. Scale bar, 40 μm. (B) Effect of paeoniflorin on body size of C. elegans. Wild-type nematodes are treated with paeoniflorin at the indicated concentrations, and their TOF and EXT in a COPAS Biosort system are measured (n > 100 animals per group). Adapted from original data that support the findings in Ai et al. (2018). AU, arbitrary units.

Conservation of key nutritional pathways in C. elegans

To maximize health and survival, animals must maintain a nutritional balance in line with nutrient availability, which may activate a number of signal transduction networks to modulate various biochemical processes of fundamental importance, including metabolism, stress response, aging and disease (Chantranupong, Wolfson, and Sabatini 2015; Efeyan, Comb, and Sabatini 2015; Templeman and Murphy 2018). Many of these signaling cascades are highly conserved across animal kingdom, including the nematode model C. elegans (Rashid et al. 2020; Rubio-Aliaga 2012). For example, the phylogenetically conserved insulin/insulin-like growth factor (IGF) signaling (IIS) pathway is known to play pivotal roles in dietary response, metabolic control and lifespan determination. The main signaling components of this pathway in C. elegans are DAF-2, AGE-1, AKT and DAF-16, which are homologous to insulin/IGF receptor (IGFR), phosphoinositide 3-kinase (PI3K), protein kinase B (PKB) and forkhead box O (FOXO) transcription factor, respectively, in humans (López-Otín et al. 2013; Xiao et al. 2022; Zhang et al. 2012).

The AMP-activated protein kinase (AMPK), a serine/threonine protein kinase ubiquitously expressed in eukaryotic cells, is a critical sensor of energy status that controls the response of several energy-related pathways to energy depletion, nutritional cues and other signals (Herzig and Shaw 2018; López et al. 2016). As the energy gauge of eukaryotes, AMPK plays an important role in cellular metabolism and metabolic diseases, including uptake and metabolism of sugars and fatty acids (Day, Ford, and Steinberg 2017; Herzig and Shaw 2018; Kahn et al. 2005). Another serine/threonine protein kinase known as target of rapamycin (TOR) or mammalian/mechanistic TOR (mTOR) is also a major nutrient sensor evolutionarily conserved in eukaryotes (Chantranupong, Wolfson, and Sabatini 2015). TOR/mTOR is known to play an integral role in response to signals from IIS and AMPK as well as nutrients (e.g. leucine and arginine), and serves as a master regulator of metabolic homeostasis and cell growth (Blackwell et al. 2019; Kim and Guan 2019; Saxton and Sabatini 2017).

Another well-characterized pathway linked to nutrient signaling involves the mitogen-activated protein kinases (MAPK), including c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinases (ERK) and p38 kinases, which are widespread in eukaryotes from invertebrates to mammals and have diverse physiological functions such as stress and metabolic responses (Darling and Cook 2014; Huang and Tunnacliffe 2004; Huang, Banton, and Tunnacliffe 2010; Nikolic, Leiva, and Sabio 2020). Phylogenetic and functional analysis of C. elegans has identified four JNK homologs (JNK-1, KGB-1, KGB-2 and C49C3.10), two ERK homologs (MPK-1 for ERK1/2 and MPK-2 for the rare ERK5) and three p38 MAPK homologs (PMK-1, PMK-2 and PMK-3) (Andrusiak and Jin 2016; Heger et al. 2010; Lackner et al. 1994), providing invaluable models to study MAPK associated nutrition signalings as demonstrated for peptides, polysaccharides and other functional food components (Li et al. 2017; Xiao et al. 2022; Yoon et al. 2019).

Many other signal transduction pathways of nutritional and metabolic importance are also conserved in C. elegans. For example, the lifespan-extending effect of caloric restriction, an extensively studied dietary regimen across species, has been well established in C. elegans. The highly conserved molecular signature of dietary restriction is found not only closely associated with the aforementioned IIS and TOR/mTOR pathways but also related to other conserved signaling cascades such as the NAD-dependent protein deacetylase SIR-2.1 (homologous to human SIRT1) and the stress response transcription factor SKN-1 (homolog of human Nrf2) as well as conserved epigenetic mechanisms (Bishop and Guarente 2007; Hernández-Saavedra et al. 2019; Kapahi, Kaeberlein, and Hansen 2017; Lakowski and Hekimi 1998). Taken together, the strong conservation of these signaling principles and the extensive experimental investigations in C. elegans make this invertebrate animal a convenient yet elegant in vivo model for nutrition biology studies and also a highly promising tool to provide further insights into molecular mechanisms of human nutrition.

Perception and consumption of food in C. elegans

Food consumption is essential for animals and humans to maintain physiological integrity and live in a healthy state, but food supplements may have different impacts on their behaviors, including feeding performance. C. elegans has a number of conserved food-intake regulation mechanisms orthologous to humans, and thus has been used as a model system to study feeding behaviors and phenotypes (Gomez-Amaro et al. 2015; Rodríguez-Palero et al. 2018). When they encounter unfavorable or harmful food, their food intake will be reduced, which may affect their survival and fecundity and result in an altered food clearance rate in a short period of time (Voisine et al. 2007). Remarkably, C. elegans has been shown to exhibit dietary choice for “good” food and “bad” food through mechanisms such as pheromones (Greene et al. 2016; Shtonda and Avery 2006), and is recently found even to discriminate colors to guide their foraging decisions for bacterial food (Ghosh et al. 2021).

The perception of food has evolved as one of the essential sensory functions in animals, and has been the subject of considerable studies in animals and humans. An example methodology to study food perception in C. elegans is illustrated in Figure 2, which shows that either binary choice assays or multiple choice experiments can be used to determine food preference, including differentiation of spiked compounds as well as pathogenic bacteria in food (Zhang, Lu, and Bargmann 2005). Both tests can be easily performed in a single assay plate, and food preference can be expressed as choice index (Figure 2A) or choice percentage (Figure 2B). Thanks to its sensitivity to a variety of different substances such as food additives, pesticides, heavy metals and toxins, C. elegans has been increasingly used as an alternative model animal to test the efficacy and safety of food ingredients, especially in early and rapid assessments (Ma et al. 2019; M. Zhang, Li, et al. 2019; Zhang et al. 2021). For example, C. elegans has been used as a model in nutrition research to detect various neurodevelopmental toxins (Hunt et al. 2018).

Figure 2. Experimental design for food choice assay in Caenorhabditis elegans. (A) Binary choice assay. Food selectivity can be assessed by measuring the olfactory preference of C. elegans. The test plate is divided into two different food zones, e.g. a zone seeded with E. coli OP50 as control food and a zone of E. coli OP50 with supplements as test food. Approximately 100–200 animals are placed at the center of the plate, allowed to move freely for a period of time (e.g. 10 h at 20 °C), and scored in each zone. A choice index for a food is calculated according to the equation shown, where an index of 1.0, -1.0 and 0 indicates complete preference, complete avoidance and no preference of the food, respectively. (B) Multiple choice assay. In multiple choice experiments, the test plate is divided into several zones for different foods. The animals are placed at the center of the plate, roughly equidistant from each zone, allowed to move, and scored in each zone as in (A). Food preference is expressed as a percentage of animals in the food zone relative to total animals.

Sugar status and metabolic reprogramming in C. elegans

Animals and humans primarily use sugars to produce energy, but different amounts and types consumed may have profoundly different consequences despite undisputed importance of sugars in health (Clemens et al. 2016). For example, high glucose has been found to shorten the lifespan of C. elegans (Lee, Murphy, and Kenyon 2009; Schlotterer et al. 2009), while glucose restriction (Schulz et al. 2007) and trehalose supplementation (Honda, Tanaka, and Honda 2010) are shown to extend its lifespan. When fed with high-glucose diets, the biochemical and physiological changes of C. elegans are indeed prominent, including lifespan, stress resistance, reproduction, body size, fat content, and glycogen storage (see examples listed in Table 1).

It has been established that the insulin signaling pathway, i.e. the DAF-2/IGFR–AGE-1/PI3K–AKT/PKB–DAF-16/FOXO axis, plays an important role in the response and adaptation of C. elegans to different type and different amount of sugars, including remodeling of carbohydrate and lipid metabolisms (Depuydt et al. 2014; Forsythe et al. 2006; Hanover et al. 2005; Hibshman et al. 2017; Seo et al. 2018). For example, triglycerides are the main energy storage in wild-type (N2) C. elegans under normal conditions, while glycogen storage and use are increased in daf-2 mutants (Seo et al. 2018). When glycogen is suppressed, trehalose storage is increased. Interestingly, the lifespan and healthspan of C. elegans are increased upon this metabolic shift from glycogen to trehalose (Seo et al. 2018). In particular, the metabolic shift between glycogen and trehalose is shown to play a part in development and stress responses, including oxidative stress and starvation, via the DAF-2–DAF-16 insulin pathway (Gusarov et al. 2017; Hibshman et al. 2017; Penkov et al. 2020).

Lifetime and transgenerational nutrition tracking in C. elegans

Due to nutritional memory, early-life dietary intervention may affect the health and fitness outcomes of animals and humans later in life even after resuming normal diet, and this may also have an impact on their progeny (Hahn et al. 2019; Jobson et al. 2015; Mautz, Lind, and Maklakov 2020). With a short life cycle and lifespan, C. elegans can be easily exposed to different dietary conditions at any particular stage of development and monitor its nutritional response for a specific period of time, including lifelong and cross-generational tracking. A notable example is the above-mentioned adverse effects of high glucose, which is the major energy source and also a key regulator of metabolism. Some of these effects are heritable in C. elegans as transgenerational phenotypes are observed in the next generations of progeny despite only the first generation being exposed to high glucose (Alcántar-Fernández et al. 2018, 2019; Tauffenberger and Parker 2014). Another example is the studies on starvation throughout the life cycle of C. elegans as recently reviewed (Baugh and Hu 2020). Extended period of food deprivation at larval stages is found to have long-term phenotypic consequences on growth, development, reproduction and survival in later life and also induces transgenerational inheritance (Jobson et al. 2015; Jordan et al. 2019; Rechavi et al. 2014; Webster et al. 2018).

While starvation is basically a prolonged food deprivation with adverse consequences, dietary restriction is in essence a measured starvation resulting in low caloric intake but avoiding malnutrition, which is known to promote lifespan extension across diverse species, including C. elegans, Drosophila and mouse. However, this caloric intervention paradigm is recently shown to improve the organismal fitness of C. elegans in later life but reduce the fitness of their progeny, indicating an intergenerational tradeoff of fitness between parent and progeny nematodes (Mautz, Lind, and Maklakov 2020). On the other hand, the descendants of severely starved larvae are shown to be more resistant to starvation and stress than that of the nematodes not starved in the first generation (Jobson et al. 2015; Jordan et al. 2019). The transgenerational inheritance of these starvation phenotypes is found to be mediated by small RNA molecules, which act on genes associated with nutrient-response genes (Rechavi et al. 2014), and vitellogenin-mediated intergenerational transmission of IIS-to-IIS signals, which convey adaptation to diet availability (Jordan et al. 2019). Interestingly, the link between glucose metabolism and epigenetic signaling has also been established, which is likely to be mediated by the glucose-derived metabolite acetyl-CoA (Wellen et al. 2009). Collectively, these studies support transgenerational memory of ancestral dietary conditions and also adaptive modulation of epigenetic machinery by nutrition.

C. elegans as a simplified model for studies of diet–microbiota–host interactions

It is well recognized that health and disease are under the direct influence of microbial communities associated with humans and animals (Fontana and Partridge 2015; Leulier et al. 2017; Suzuki and Ley 2020; The Integrative HMP (iHMP) Research Network Consortium 2019). For example, gut microbiota is found to be a critical determinant of animal physiology and as a whole is linked to many aspects of host health. However, the functional interactions between food ingredients, which are the key regulators of host as well as microbial physiology, and gut microbiota (including their metabolites) still remain to be elucidated. In particular, further dissection of the intricate yet robust relationships between gut microbes and host nutrition following a more holistic approach would reveal the dynamic roles of nutrients in balancing gut microbiota and host metabolism, providing a comprehensive insight into microbial management in health and disease.

Until now, most research involving C. elegans has been performed on nematodes fed with standard monoxenic bacterial diet, usually E. coli strain OP50 on solid NGM plates and strain NA22 or HB101 in liquid culture (Figure 3), essentially in the absence of other bacteria. This type of C. elegans model does have an advantage in testing the direct functions and mechanisms of food ingredients. Since both C. elegans and E. coli are well-characterized model organisms, this monoxenic pattern of host with single-species bacteria can also serve as a simplistic but tractable “microbiota” model, in which the standard OP50 bacteria can be easily substituted by or cocultivated with other bacteria (Cabreiro and Gems 2013; Calvo et al. 2016; Gerbaba, Green-Harrison, and Buret 2017; Ortiz de Ora and Bess 2021). For example, C. elegans has shown significant metabolic, defense and transcriptomic response changes when grown at different temperatures (15, 20 and 25 °C), but distinct patterns of response to temperatures are observed when its food is changed from the standard E. coli diet to Bacillus subtilis (Gómez-Orte et al. 2018). Interestingly, the lifespan of C. elegans has been reported to be greater when fed on B. subtilis than on E. coli because of the differences in bacterially-derived metabolites such as nitric oxide and coenzyme Q, which may affect longevity-associated signaling pathways such as redox homeostasis, HSF-1 and DAF-16 (Garsin et al. 2003; Gusarov et al. 2013; Sánchez-Blanco et al. 2016).

Figure 3. Growth of Caenorhabditis elegans at different scales and throughputs. (A) Laboratory maintenance and standard culture of C. elegans. Routine culture of C. elegans is maintained on NGM agar plates seeded with a lawn of E. coli OP50 as food source. The nematodes are usually grown at 20 °C unless otherwise specified. (B) Mass culture of C. elegans on peptone-rich or egg yolk-enriched solid NGM. To obtain biochemical or larger quantities, C. elegans can be grown in mass cultures using peptone-rich or chicken egg yolk-enriched NGM trays seeded with a thick layer of E. coli NA22 as food. (C) Large-scale growth of C. elegans in liquid culture. Large quantities of nematodes can be cultured in flasks up to liters and decaliters using S medium with E. coli NA22 (or HB101) as food source or axenic liquid nematode medium. (D) Liquid culture of C. elegans for high-throughput investigations. C. elegans can be grown in 96- or 384-well plates with S medium and bacterial food or with axenic liquid nematode medium, and thus can be conveniently used in high-throughput investigations of nutritionally and pharmacologically important compounds.

Like in humans, microbial communities associated with C. elegans are also shown to play a critical role in its physiology, making it a promising model for mechanistic studies of gut microbiota (Dirksen et al. 2020; Gerbaba, Green-Harrison, and Buret 2017; O’Donnell et al. 2020; Shapira 2017; Zhang, Holdorf, et al. 2017). For example, multiple species of commensal Providencia bacteria are found to colonize the gut and alter the olfactory behavior of C. elegans, promoting fitness of both the host and the bacteria (O’Donnell et al. 2020). Further investigation suggests that the sensory alteration is caused by the bacterially produced tyramine, which is a neuromodulator that can be used by C. elegans to modulate olfactory response via ASH nociceptive neurons, demonstrating a gut–microbiota–brain signaling in the nematodes (O’Donnell et al. 2020). On the other hand, a consortium of foodborne lactic acid bacteria has been used to feed C. elegans and different bacteria are found to have differential gut colonization capacities (Zanni et al. 2015). Intriguingly, supplementation of the bacterial consortium is shown to decrease lifespan, reduce brood size, accumulate lipid droplets, and alter expression of obesity-associated genes such as nhr-49, pept-1 and tub-1, as compared to the nematodes fed the standard E. coli diet or probiotic bacterial strain (Zanni et al. 2015).

Unlike the monoxenically fed C. elegans, however, their wild counterparts are found to harbor a microbial community of diverse species in the gut. Interestingly, the gut microflora is conserved in wild C. elegans from various natural environments of distinct geographical locations, which appears to serve as a core microbiota beneficial to the nematodes, including fitness and adaptation (Berg et al. 2016; Dirksen et al. 2016; Samuel et al. 2016; Schulenburg and Félix 2017). Based on these investigations of natural microflora, CeMbio, a simplified model of C. elegans microbiota, has been recently established and become publicly available (Dirksen et al. 2020). CeMbio includes a set of 12 bacteria of different families, representing the core microbiota taxa of natural environment (Dirksen et al. 2020). Such a gnotobiotic model of C. elegans with native microbiota will undoubtedly help understand nematode biology, including metabolism, immunity, fertility, aging and gut–brain axis (Dirksen et al. 2020; O’Donnell et al. 2020; Ortiz de Ora and Bess 2021; Shapira 2017). Together with the small size and genetic tractability of C. elegans host, this type of simplified model is expected to become commonplace in microbiota studies, including high-throughput investigations of dynamic diet–microbiota–host interactions, which is expected to advance our understanding of how dietary nutrients are integrated by microbiota and host to coordinate nutrient homeostasis and thus affect health and disease.

In summary, many cutting-edge technologies originated from or developed in C. elegans, e.g. genome-wide RNA interference (RNAi), have revolutionized biomedical research in general and are increasingly contributing to nutritional biology in particular. Indeed, as a versatile and affordable in vivo model, C. elegans can be conveniently and effectively used in almost every aspect of nutrition science at molecular, cellular and organismal levels, e.g. integrated investigation on the nutritional modulation of gut-brain axis.

Limitations and solutions of using C. elegans as a food and nutrition model

As exemplified above and reported in an increasing body of literature (e.g. those listed in Table 1), significant progress in food and nutrition studies has been made using C. elegans models. We envision that continued innovation in technology combined with the inherent strengths of C. elegans model, such as genome-editing, genome-wide association study (GWAS), in vivo high-throughput, in vivo microfluidic, and automatic lifetime behavioral tracking technologies, will drive further development in food and nutrition research. However, a number of issues related to nutritional studies need to be adequately addressed before we can bring the well-established knowledge and leading-edge technologies into full play. Pho and MacNeil (2019) have recently discussed the potential causes of experimental variability in C. elegans biology between laboratories and between experiments, including variation of bacterial diet, inconsistency of growth media and genetic background of animal population per se. Here we focus on laboratory cultivation and dietary standardization with an emphasis on feeding mode and growth scales.

Culture of bacteria-fed C. elegans at different scales and throughputs

In wild, C. elegans is a nematode known to prey conservatively but selectively on bacteria as mentioned above. In laboratory, feeding C. elegans with the uracil auxotroph E. coli OP50 on solid NGM plates has become a canonical protocol for maintenance and most experiments (Figure 3A)—growth of OP50 on NGM is limited, allowing easier handling and observation of the nematodes (Brenner 1974). This cost-effective cultivation is typically carried out in 9 cm petri dishes, but 3.5 cm and 15 cm plates are also used depending on specific experiments. However, when large quantities of nematodes become necessary, the routine NGM culture plates may not suffice to meet the need. To this end, C. elegans can be grown in big trays using peptone-rich or chicken egg yolk-enriched solid NGM seeded with the E. coli NA22 as food, growing in thick layers (Figure 3B) (Miedel et al. 2014; Strange, Christensen, and Morrison 2007). Alternatively, large-scale growth of C. elegans can be achieved in liquid culture using S medium and E. coli strain NA22 or HB101 as food, which can be performed in flasks at the scale of liters and decaliters. To avoid food and oxygen shortages, the culture is gently but continuously aerated with chemically or filter sterilized ambient air (Figure 3C), and more bacteria can be added before they are depleted to ensure food supply. Axenic liquid nematode medium can also be used in large-scale culture of C. elegans, but further development and practice are needed.

In addition to growing nematodes in large quantities, liquid culture has also become increasingly common in laboratory for better control of dietary supplements, including nutritionally and pharmacologically important compounds (Hibshman, Webster, and Baugh 2021; Lev et al. 2019). In particular, C. elegans can be grown in liquid culture with S medium in 96- or 384-well plates if high-throughput investigation is to be performed (Figure 3D). Particular attention should be paid to the amount of food, the density of nematodes, and the total volume per well to ensure survival and avoid dietary restriction of the animals (Wang et al. 2021). In addition to bacterial feeding, axenic liquid culture of C. elegans is also a promising alternative that can be used in high-throughput investigations.

Uptake of dietary supplements in bacteria-fed C. elegans culture

Caenorhabditis elegans is known to obtain compounds from its surroundings through three routes, i.e. ingestion by the pharynx, absorption via the skin-like cuticle, and uptake through the exposed neuronal endings (Kaletta and Hengartner 2006). In practice, supplementation of functional ingredients, including nutrients and therapeutics, to bacteria-fed C. elegans can be done by adding to the medium (e.g. NGM or S Medium), directly spreading on the NGM plates, or mixing with the bacterial food and then spreading on the NGM plates. For convenience, many studies directly add supplements into the NGM agar medium or the liquid S Medium (e.g. Koseki et al. 2020). In the case of NGM supplementation, however, it is difficult to know how much of the supplemented compounds is taken by the nematodes. It may end up with very high “doses” to show desired nutritional effects particularly if the supplements are inside the NGM agar, while high concentrations may on the other hand cause osmotic pressure, affecting the outcomes of experiments (Koseki et al. 2020; Lublin and Link 2013; Zheng et al. 2013).

Whilst nutrients added in the liquid medium can be uptaken by ingestion through the pharynx, the supplements added inside NGM agar are likely to be taken by absorption via cuticle and exposed neuronal cilia rather than by direct pharyngeal ingestion, particularly for small-molecule compounds. This is, in the common sense, not “food” intake and ingestion but may nevertheless still be misinterpreted as “dietary” data. In this regard, supplementation of E. coli OP50 food with functional ingredients is a better option. Yet, metabolic interference from the bacteria may preclude direct examination on the nutrients of interest. For this purpose, the live bacterial food can be replaced with dead OP50 E. coli killed by UV irradiation or heat (e.g. Cabreiro et al. 2013; Seo et al. 2018; Wood et al. 2004). However, special attention should be paid to the preparation of UV- and, in particular, heat-killed bacteria so that essential nutrients are retained in the bacterial food; otherwise dietary restriction may arise.

Distinct effects of different bacterial strains as food on C. elegans

As indicated above, wild C. elegans forages for a variety of bacteria as food in its natural habitat, and different bacterial food may have distinct effects on the nematodes, including fitness, lifespan, reproduction and stress response (e.g. Coolon et al. 2009). These findings suggest that there may exist differential nutrients and bioactive metabolites in the bacteria. Under laboratory conditions, C. elegans is usually cultured on NGM agar plates seeded with a lawn of standardized E. coli bacteria, whose major macronutrient is proteins. On average, the contents of proteins, lipids and carbohydrates in E. coli cells are 55%, 7–9% and 6% respectively by dry weight, i.e. a macronutrient ratio (%) of approximately 80:10:10 (protein:fat:carbohydrate), and the overall mass levels of proteins and carbohydrates of E. coli food are roughly similar to that of C. elegans as well as mammals (Zečić, Dhondt, and Braeckman 2019). The content of micronutrients, including vitamins and minerals, accounts for ∼4% of bacterial dry weight, and different E. coli strains contain different levels of dietary micronutrients (e.g. folate and vitamins) to C. elegans (Zečić, Dhondt, and Braeckman 2019).

The macronutrients of E. coli strains commonly used as C. elegans food source in laboratory are roughly similar but with different carbohydrate contents and fatty acid compositions. For example, similar levels of proteins and fatty acids are found in the standard laboratory strain OP50 (a uracil auxotroph derived from the standard B strain), the RNAi feeding strain HT115 (an RNase III-deficient strain derived from K12 strain) and another popular laboratory E. coli strain HB101 (a hybrid of strains B and K12), but the content of total carbohydrates in OP50 is 3–5 times less than that in HT115 and HB101 strains (Brooks, Liang, and Watts 2009; MacNeil et al. 2013). HB101 can form thicker lawns than OP50 on NGM plates and is recommended for liquid cultures of C. elegans (Brooks, Liang, and Watts 2009; Hibshman, Webster, and Baugh 2021; MacNeil et al. 2013).

Interestingly, almost no impact is found on the lifespan of wild-type C. elegans grown on these E. coli strains (Brooks, Liang, and Watts 2009; Pang and Curran 2014; Soukas et al. 2009). HB101 is, however, considered to be a high-quality food due to its capability to provide better sustenance for growth rate, body size and brood size of C. elegans as compared to OP50 and OP50-derived strains (Avery and Shtonda 2003; Shtonda and Avery 2006; So, Miyahara, and Ohshima 2011; Sowa et al. 2015). While the phospholipid levels of C. elegans feeding on these E. coli strains are similar, the triacylglycerol level of OP50-fed nematodes is almost twice the amount of that in nematodes fed with HT115 or HB101 bacteria (Brooks, Liang, and Watts 2009), demonstrating potential reprogramming of metabolic pathways leading to metabolic adaptation of different diets. Therefore, control food settings are of paramount importance in any experiments involving bacteria-fed C. elegans.

Chemically defined medium for C. elegans culture

Dietary composition is undoubtedly a major player in animal physiology and health, and C. elegans is known to adapt to a variety of bacterial diets likely through reprogramming of metabolic pathways as aforementioned (e.g. Pang and Curran 2014; Watson et al. 2013). Therefore, potential metabolism of supplemented nutrients by the bacteria may contribute, for good or for bad, to the nutritional and pharmacological effects of the supplements under investigation, which may well be intended only to study the direct food–animal interactions. To this end and also for convenience, attempts have been made to develop defined axenic media for C. elegans culture (Lu and Goetsch 1993; Szewczyk, Kozak, and Conley 2003; Vanfleteren 1976), e.g. CeMM (C. elegans Maintenance Medium) and CeHR (C. elegans Habitation and Reproduction) media (Table 2), excluding bacterial metabolism-associated variables in nutrition study. It is advisable to make the chemically defined media from a series of component preparations, including vitamins and growth factor solutions, salt solutions, amino acids, nucleic acid substituents, other growth factors, and energy source (either D-glucose or potassium acetate) (Szewczyk, Kozak, and Conley 2003). Most of these component groups can be prepared in advance and stored frozen for up to six months. Upon use, the medium is compounded with the solutions in proportion as detailed in the supplementary protocol of Szewczyk, Kozak, and Conley (2003).

Table 2. Comparison of commonly used culture methods of C. elegans.

Food/medium	Ingredients	Lifespan	Pros	Cons
E. coli OP50 (NGM, solid)	Bacteria	25 days	Regarded as gold standard (particularly for lifespan)	Potential bacterial metabolism
E. coli NA22 (S Medium, liquid)	Bacteria	25 days	Accepted as standard in laboratory; high throughput screens	Potential bacterial metabolism
AXM (Liquid)	Soy-peptone; dry yeast extract; hemoglobin (Lenaerts et al. 2008)	25 days	Convenient preparation; high throughput screens; no bacterial metabolism	Dietary restriction; altered growth, reproduction and metabolism (vs NGM)^a
CeMM (Liquid)	Vitamins; growth factors; amino acids; nucleic acid substituents; salts (Lu and Goetsch 1993)	60 days	Clear chemical composition; high throughput screens; no bacterial metabolism	Complex preparation; altered growth, reproduction and metabolism (vs NGM)^a
CeHR (Liquid)	CeMM + 20% milk (Flavel et al. 2018)	60 days	Better growth (vs CeMM); high throughput screens; no bacterial metabolism	Complex preparation; altered growth, reproduction and metabolism (vs NGM)^a

^aIncluding delay of growth, reduction of reproduction, decrease of lipid and protein storage, and alteration of energy metabolism gene expression (Castelein et al. 2008; Lenaerts et al. 2008; Szewczyk et al. 2006; Zhang et al. 2015).

Abbreviations: AXM, axenic medium; CeHR, C. elegans habitation and reproduction (medium); CeMM, C. elegans maintenance medium; NGM, nematode growth medium.

Axenic culture of C. elegans in chemically defined liquid medium

Caenorhabditis elegans is often described as a free-living soil nematode but is typically found in rotting vegetation rather than in “pure” soil, and as a known bacterivore it may also take in plant or animal materials and yeast cells (Félix and Duveau 2012; Frézal and Félix 2015; Schulenburg and Félix 2017; Zečić, Dhondt, and Braeckman 2019). Therefore, various extracts, including those of yeast, chick embryo and liver, were included in early attempts of bacteria-free culture of C. elegans to ensure its nutritional requirements. Based on nutritional framework borrowed from E. coli diet (e.g. amino acid ratio), CbMM (Caenorhabditis briggsae Maintenance Medium) was developed as a nematode basal medium, which contains glucose, amino acids, vitamins, minerals and growth factors (Buecher, Hansen, and Yarwood 1966; Sayre, Hansen, and Yarwood 1963), and later modified as CeMM, a chemically defined axenic medium (Table 2), by adding potassium acetate or extra glucose as energy source (Lu and Goetsch 1993; Szewczyk, Kozak, and Conley 2003). Another bacteria-free medium is CeHR (Table 2), which is similar to CeMM but supplemented with either 10% or 20% milk to alleviate growth, development and fecundity of C. elegans altered in axenic liquid culture media (Flavel et al. 2018; Rao et al. 2005). The addition of milk, however, makes CeHR no longer a chemically defined medium in a strict sense.

Take CeMM as an example of defined medium, every nutrient added to the medium is quantified and each factor can be individually studied without potential interference of bacteria (Szewczyk, Kozak, and Conley 2003), e.g. in the discovery of novel bioactive monosaccharides (Sakoguchi et al. 2016a, 2016b). Furthermore, CeMM has been demonstrated to be better suited than bacterial food for automatic detection and long-term tracking of C. elegans, e.g. in sophisticated worm microfluidics avoiding pipeline blockage caused by bacterial aggregation (Doh et al. 2016), in long-term recordings of locomotor activity using infrared microbeam scattering without frequent worm transfer (Simonetta and Golombek 2007), or in spaceflight-related experiments (Adenle, Johnsen, and Szewczyk 2009; Qiao et al. 2013; Tee et al. 2017).

Adaptation of C. elegans in chemically defined liquid medium

Normal movement of C. elegans requires a layer of water surrounding its body surface. In natural habitat as well as on the standard NGM agar plates, C. elegans lives near the liquid–air interface, making it rapidly adaptable to the two distinct physical conditions. For example, the pattern of locomotion can switch quickly between crawling and swimming movements driven by S- and C-shaped body kinematics typical for solid and liquid cultures, respectively (Korta et al. 2007; Pierce-Shimomura et al. 2008). Therefore, one of the major adaptations of C. elegans to axenic liquid medium is the long-term impact of aquatic environment to their movement kinematics. In addition to behavioral changes, morphological, physiological and transcriptional differences may also arise when C. elegans is grown in liquid culture as compared to culture on agar plates (Çelen, Doh, and Sabanayagam 2018; Hibshman, Webster, and Baugh 2021; Houthoofd et al. 2002; Lev et al. 2019; Szewczyk, Kozak, and Conley 2003; Szewczyk et al. 2006). For instance, a number of differentially expressed genes in the axenic liquid media (including CeMM and CeHR) are found to correlate with the observed phenotypes (e.g. body morphology and cuticle development), highlighting the importance to standardize controls when switch between different culture media (Çelen, Doh, and Sabanayagam 2018; Pierce-Shimomura et al. 2008).

Sensory perception of food is essential for C. elegans to survive in microbe-rich natural habitats. As a powerful model organism in genetics and neurobiology, the functions of olfactory neurons of C. elegans are well established, including food search and aversive responses (e.g. avoidance of pathogens), and the sensory transduction pathways (e.g. Ras-MAPK) are conserved with higher animals (Hirotsu and Iino 2005). Interestingly, the olfactory neurons have been found to respond to a number of chemical cues produced specifically by bacteria (e.g. Bargmann, Hartwieg, and Horvitz 1993; O’Donnell et al. 2020; Zhang, Lu, and Bargmann 2005). Therefore, another adaptation of C. elegans to axenic media is the lack of potentially influential chemicals produced by the bacterial food source, e.g. the absence of bacteria may decrease the expression of sensory signaling-associated genes in nematodes grown in CeMM (Mukhopadhyay et al. 2008). Taken together, C. elegans shows a biological instinct to trigger molecular control mechanism underlying its robust phenotypic plasticity in response to changes in environment and nutrient.

Impact of particles on C. elegans in axenic liquid culture

It is well-known that the feeding of C. elegans depends on the rhythmic contraction of the pharynx, a neuromuscular organ that consists of 20 neurons and 20 muscle cells and functions as a pump connecting the mouth to the intestine (Figure 1A). Of the neurons, the MC cholinergic motoneurons are controlled by food sensation (mechanosensation) and serotonin signal and directly linked to food pumping dynamics (Avery and You 2012; Franks et al. 2006). The pharyngeal pumping rate is found to vary in response to food availability, at ∼1 pump/sec without food but ∼4 pumps/sec in the presence of bacterial food, which is considered to arise from the stimulation of the pharynx by increased bacterial particles (Franks et al. 2006; Lee et al. 2017). In wild-type C. elegans, MC neurons are key cholinergic neurons of the pharynx, which are associated with response to nutrient changes and pumping rate through acetylcholine secretion (Ishita, Chihara, and Okumura 2020; Zhang et al. 2015). Interestingly, signalings from the pharyngeal MC neurons and body wall muscles, including eat-2 (encoding an acetylcholine receptor) and cha-1 (encoding an acetylcholine synthetic enzyme), are shown to regulate the growth and development of nematodes grown in CeMM medium, suggesting the involvement of mechanical intake of nutrients by pharynx (Zhang et al. 2015).

CeMM is a full nematode medium with an excess of basic nutrients. Intriguingly, however, C. elegans cultured in this medium still shows suboptimal growth, a state between standard E. coli OP50 diet and dietary restriction-like phenotypes, suggesting poor nutrient intake or utilization, despite the fact that the nematodes are “healthy” in term of full growth cycle (Castelein et al. 2008; Lenaerts et al. 2008; Szewczyk et al. 2006; Zhang et al. 2015). On the other hand, apart from nutritional effects, the added milk in CeHR is also considered to provide the medium with particulate matter, which is required by C. elegans to help take up the nutrients in the liquid medium (Flavel et al. 2018; Zečić, Dhondt, and Braeckman 2019). The growth-promoting effect of added yeast extract, e.g. in AXM medium (Table 2), as well as some pure precipitated proteins may also arise at least partially from their capacity to form particulates (Zečić, Dhondt, and Braeckman 2019). As a matter of fact, food uptake in the nematodes is indeed mediated by the pharynx which filters food particulates from liquid prior to pumping to the intestinal system (Zečić, Dhondt, and Braeckman 2019), and thus stimulation of the pharynx by the particles is likely involved in promoting nutrient uptake in C. elegans.

Development of axenic NemaFood microgranules for C. elegans

As discussed above, the use of defined liquid medium is satisfactorily effective in avoiding potential metabolic interference by bacteria in food and nutrition studies, but the added nutrients are absorbed not only through the pharynx, i.e. as food “eaten,” but also through the epidermis, which in a strict sense is not food. Even in the case of pharynx-mediated feeding, lack of particles in the chemically defined liquid medium may also have negative effects on food uptake and consequently physiological status. On the other hand, nanoparticles and nanoemulsions have been successfully used as delivery vehicles to bring drugs and nutrients into the digestive system of C. elegans (Colmenares et al. 2016; Lucio et al. 2017, 2018; Shen et al. 2019). Therefore, we have microgranulated the axenic liquid medium into nano- and micron-scale particles for use as nematode food (“NemaFood”) (Huang, Xiao, et al. 2022). The fabrication of NemaFood submicrogranules is performed by using edible polymers, e.g. formulation of the axenic liquid medium AXM (Table 2) with quaternized chitosan and alginate using the method originally intended for oral delivery of proteins in other systems (Li et al. 2007).

The NemaFood is used to feed C. elegans on NGM agar plate in the same fashion as the OP50 bacterial food. As shown in Figure 4, the semi-defined AXM-based NemaFood appears sufficient to support the growth of C. elegans. For instance, the body shape and behavior of NemaFood-fed animals are virtually comparable with the OP50-fed counterparts on NGM solid plates, significantly superior to those grown in S. medium with NA22 bacteria or in liquid AXM. These results demonstrate, as a proof of concept, the feasibility of nano/microparticulate NemaFood, either it be prepared from chemically defined medium, semi-defined nutrients, or killed bacteria. In particular, it is promising to standardize C. elegans base diet using microparticulated holidic food comprising of defined macro- and micronutrients, which mimics not only the nutrient composition but also morphology of bacterial food and thus provides a better platform for nutritional and pharmacological research in C. elegans.

Figure 4. Axenic culture of Caenorhabditis elegans using liquid medium and microgranulated NemaFood. Representative images are shown for wild-type C. elegans grown in axenic liquid AXM medium (A) and by feeding AXM–chitosan–alginate microparticles (“NemaFood”) on solid NGM plates (B); for comparison, the nematodes grown in liquid S medium with NA22 bacteria as food (C) and by feeding OP50 bacteria on solid NGM plates (D) are also shown. Scale bar, 100 μm. The AXM liquid medium is prepared as described previously (Table 2; Lenaerts et al. 2008). The AXM-based NemaFood microgranules are prepared by microgranulating the AXM liquid medium into nano- and micron-scale particles with edible polymers, as exemplified here by quaternized chitosan and sodium alginate using previously described method for oral delivery of nutrients (Li et al. 2007).

Conclusions

Caenorhabditis elegans has been used as a model animal for half a century in life sciences, and emerging evidence has begun to suggest its transformative impact on nutrition biology. The present article attempts to assemble the current knowledge on C. elegans as a nutrition model and draw a clearer picture of the tradeoffs between its strengths and limitations. Although the experimentally tractable nematode is small in size, it does have various tissues and organs, including nervous, muscle, intestinal and reproductive systems, and its key nutritional pathways and disease genes are highly conserved with humans. For example, C. elegans is known to have well conserved food-intake regulation mechanisms and displays food perception and preference. Alongside conventional culture on solid plates seeded with bacteria, C. elegans is increasingly grown in liquid culture with bacterial food at different scales and throughputs, from microliters in 384-well plates to decaliters in culture tanks. In addition, C. elegans can also grow axenically in chemically defined liquid medium, avoiding potential metabolic interference by bacteria. However, axenically grown and indeed even bacteria-fed C. elegans in liquid culture shows somewhat suboptimal, albeit healthy, growth as compared to bacteria-fed counterparts on solid plates. To eliminate experimental variability, therefore, dietary standardization and feeding mode in C. elegans culture are of great importance. In a proof-of-concept attempt, axenic liquid medium is microgranulated into microparticulate nematode food (“NemaFood”) and successfully applied to C. elegans by feeding on solid plates, essentially comparable with bacterial food. In conclusion, as an affordable in vivo model, C. elegans can be conveniently used in almost every aspect of food and nutrition research at molecular, cellular and organismal levels. It is our view that one of the major challenges to unleash the groundbreaking potential of C. elegans as a nutrition model is standardization of its base diet, which is overdue as compared to other model animals such as fruit fly, zebrafish and mice.

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

This work was supported by Guangdong Province Medical Products Administration (grant numbers 2022YDZ10 and 2021ZDB15), the 111 Project (grant number B17018), and the Special Funds of the Central Finance to Support the Development of Local Universities and Colleges.