Agar-based solid culture systems are the most common
Detailed methods for lifespan assays using solid medium systems are described in another review article (Amrit et al., 2014), but here, we will discuss the methods very briefly. First, synchronization of the specific developmental stages of worms is required. Gravid adult worms are allowed to lay eggs overnight on bacteria-seeded NGM plates and are subsequently removed from the plates. Animals hatched from the eggs that become young adults are then transferred to fresh bacteria-seeded NGM plates for lifespan assays. The synchronized adult worms will lay eggs and need to be distinguished from their progeny. This synchronization method is the standard protocol for most solid culture-based survival assays described in this review.
After preparation of sufficient young-adult-stage animals, the number of dead and live worms should be regularly counted, and dead worms should be removed. A general method for determining death is counting worms that do not respond to a gentle touch with a sterilized platinum wire. Alternatively, the live status of worms can be determined by observing avoidance from a heated platinum wire located close to their heads (Amrit et al., 2014). Another method is executed by flooding many replica plates with water at each time point and counting animals responsive to a gentle touch to the surface of water with a platinum wire (Samuelson et al., 2007). In all these methods, worms are censored if they are missing, have crawled off, have burrowed, or display internal hatching or vulval rupture. However, these censored worms are still included in subsequent statistical analysis.
Media for liquid culture-based
For synchronization of worms, L1 larvae that have just hatched are transferred to liquid culture medium. Before synchronization, eggs are collected from gravid adults by using a method called bleaching, which can also be used for large-scale, solid culture lifespan assays (Amrit et al., 2014). During bleaching, gravid adults are treated with sodium hypochlorite and washed with M9 buffer, a widely used phosphate-based buffer for
Methods for identifying dead worms differ between liquid and solid culture systems. In liquid-based assays, worms that do not display spontaneous movements or respond to shaking are counted as dead. Fluorescent dyes, such as SYTOX, that stain dead worms by penetrating their cell membranes and emit fluorescent signals can be used to facilitate counting (Gill et al., 2003). An automated detector, such as a plate-reading fluorometer or COPAS BIOSORT, is used to quantify fluorescence levels over time (Gill et al., 2003; Pulak, 2006).
For both solid- and liquid-based lifespan assays, separating adult hermaphroditic worms from their progeny is a significant challenge, as progeny that have reached adulthood are difficult to distinguish from their mothers. The most straightforward way to resolve this issue is transferring synchronized adult worms for lifespan assays onto new plates every 1 or 2 days until the worms do not produce progeny. However, constant transfer of worms can cause them physical damage and is relatively labor intensive. Therefore, this method is more suitable for small-scale lifespan experiments.
For large-scale lifespan assays, several alternative and complementary methods have been used. First, treatment with 5-fluoro-2′-deoxyuridine (FUdR), which inhibits DNA synthesis, is a popular method that prevents progeny from hatching. However, caution is needed because FUdR treatment itself affects the lifespan of several mutant worms (Aitlhadj and Stürzenbaum, 2010; Rooney et al., 2014; Van Raamsdonk and Hekimi, 2011). Second, temperature-sensitive sterile mutant strains, including
Another common feature for lifespan assays is that bacteria act as food sources. Byproducts derived from bacteria may affect the lifespan of worms, and may confound results of the assays. To exclude these possibilities, worms can be fed with killed bacteria, or axenic media devoid of live food source can be used (Cabreiro et al., 2013; Lee et al., 2009; Lu and Goetsch, 1993). However, axenic media cause physiological changes in worms, including delayed development, reduced brood size, and extended lifespan (Szewczyk et al., 2006).
The conventional lifespan assays described above are manual counting assays that have several limitations; for example, manual assays are labor intensive and prone to researcher-oriented bias, and worms are vulnerable to mechanical and heat stresses induced by platinum wire usage. To overcome these limitations, various automated assay tools have been developed. For solid culture systems, “WormScan” can automatically determine the death of worms using movement parameters (Mathew et al., 2012). Subsequently, the “Lifespan Machine,” a fully automated device for measuring worm lifespan, processes time-lapse photographs and analyzes worm survival data, has been developed (Stroustrup et al., 2013). For liquid culture-based systems, a microfluidic device called “WormFarm” removes progeny based on differences in worm size and automatically detects death via video recording (Xian et al., 2013). Overall, these automated lifespan assays are appropriate for large-scale and/or unbiased assays and are complementary to the conventional manual lifespan approaches discussed above.
Research of stress resistance provides valuable information regarding the interaction between internal or external stresses and biological processes, such as cellular homeostasis. Here, we describe
Survival assays under oxidative stress conditions provide information regarding biological systems crucial for responding to oxidative stresses.
Understanding the nature of specific ROS-generating chemicals is important for properly designing oxidative stress assays (Keith et al., 2014; Sies, 1985). Paraquat, an organic compound used as an herbicide, produces superoxide anions and is widely used as an oxidative stress inducer. Hydrogen peroxide (H2O2) is a commonly used bleaching and decontaminating reagent. Tert-butyl hydroperoxide (t-BOOH) is a highly reactive and toxic organic peroxide that acts as a radical polymerization initiator. Arsenite blocks pyruvate dehydrogenase and destroys energy production systems via increasing intracellular ROS levels. Juglone is a highly poisonous organic compound that forms a semiquinone radical and induces cell death by generating a large amount of superoxide anion radicals.
Surprisingly, recent studies have shown that low concentrations of oxidative stress-generating reagents increase lifespan in many organisms, including
As oxygen is essential for aerobic organisms, animals are equipped with systems to adapt to different oxygen levels. Hypoxia and hyperoxia refer to conditions in which biosystems are exposed to abnormally low and high levels of oxygen, respectively (Rodriguez et al., 2013).
Hypoxia can be induced in two distinct ways, physical and chemical induction methods (Jiang et al., 2011; Scott et al., 2002). For physical induction, worms are placed in a sealed hypoxic chamber (< 0.2% O2) filled with a constant flow of an anoxic gas mixture containing CO2, H2, and N2. For chemical induction, worms are treated with fresh 0.5 M sodium azide, which causes cellular hypoxic responses via inhibiting mitochondrial respiration complex IV. Worms exposed to hypoxia for a specific time are then transferred to NGM agar plates for recovery in room air, and numbers of dead worms are scored at regular intervals (e.g., several hours). Hyperoxia can be achieved by using a sealed chamber filled with 60% O2 (Doonan et al., 2008). Dead worms are then counted at regular intervals (e.g., every 1 to 3 days).
High environmental temperatures cause structural and functional impairments in macromolecules, wreaking havoc on animal physiology. In
A typical temperature for acute heat stress resistance (thermotolerance) assays is 35°C (Lithgow et al., 1994). At this high temperature,
In natural environments, animals are exposed to various kinds of osmotic stresses. Hyperosmotic shock induces water efflux and protein aggregation, leading to body deterioration and protein homeostasis (proteostasis) (Choe and Strange, 2007; Rodriguez et al., 2013). Organisms are equipped with several protection mechanisms that help maintain cellular osmotic homeostasis. These include increased levels of osmoregulatory solutes that function as chemical chaperones, including glycerol, sorbitol, inositol, and trehalose, and the induction of osmoprotective genes.
Osmotic stress resistance assays using
Ultraviolet radiation (100 to 400 nm) is a major DNA-damaging environmental stress for most terrestrial organisms. UV stress induces DNA lesions and produces free radicals that damage other cellular macromolecules. Most organisms have defense mechanisms against UV radiation, such as nucleotide excision repair pathways, that repair damaged DNA (O’Neil and Rose, 2006).
Because UV radiation can cause harmful effects in humans, researchers should be cautious during UV light stress assays. Liquid media culture systems are not suitable, as liquid absorbs UV and decreases its effective dosage. Ultraviolet light has dose-dependent effects on the health and survival of
Protein homeostasis (proteostasis) is essential for cellular function and survival. Unfolded protein responses (UPRs) are elicited by many environmental stresses or genetic perturbations in the cytosol, ER, and mitochondria, and are key defense mechanisms for maintaining proteostasis. Each UPR transmits signals from a specific cellular compartment to the nucleus. Evolutionarily conserved signaling factors, including IIS components, target of rapamycin, and AMP kinase, regulate proteostatic stress responses (Vilchez et al., 2014). Among these UPRs, we will focus on ER stress assays that determine survival of animals upon treatment with chemical ER stressors that induce UPRER.
Accumulation of unfolded proteins in the ER acts as stress signals that are transmitted to the nucleus (Walter and Ron, 2011). Representative ER stress-inducing agents include tunicamycin and dithiothreitol (DTT). Tunicamycin is a widely used inhibitor of UDP-N-acetylglucosamine-dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT) (Kuo and Lampen, 1974). Therefore, tunicamycin results in accumulation of unfolded glycoproteins, which leads to ER stress. Typical
Non-essential heavy metals whose specific gravity is over 5 g/cm3, including cadmium and arsenic, have harmful effects on organisms by altering the function and structure of their proteins or by generating ROS. Bioaccumulation of heavy metals causes severe diseases in humans, such as poisoning, renal dysfunction, and damage to the central nervous system (Jaishankar et al., 2014). Molecules that detoxify heavy metals include metallothionein proteins, which contain many cysteine residues and protect cells by chelating heavy metal ions (Freedman et al., 1993). As these factors are evolutionarily well conserved, heavy metal resistance assays using
Survival assays for pathogen resistance provide important information regarding host defense mechanisms and antagonistic relationships between pathogens and hosts. Understanding pathogenesis is also the basis for identifying prophylaxis and treatment strategies against infection. Various pathogens, including bacteria, have been used for pathogen resistance assays using
Pathogenic bacteria are categorized into two groups, Gram-negative and -positive bacteria (Darby, 2005).
Among the assays described above, slow-killing assays are the most popular survival experiments using
Several specific considerations for slow-killing PA14 survival assays should be noted. First, concentrations of PA14 affect virulence of the bacteria due to quorum sensing (Papenfort and Bassler, 2016). Therefore, proper cultivation temperatures and media composition, as well as the preparation of fresh bacterial lawns, are crucial for performing reliable assays. Second, using worms in proper developmental stages is critical; young adults and L4 larvae are routinely used for the assays (Darby, 2005; Keith et al., 2014). Third, due to prevalent internal hatching of the worms, FUdR treatment is highly recommended. Fourth, dead worms should be counted every 6 to 12 hours because they quickly lyse and become transparent due to exoenzymes produced by PA14.
Opportunistic fungal pathogens, which are capable of infecting and killing
Upon completion of survival assays, data should be analyzed with proper statistical methods. Two widely used curves for survival analysis are simple survival curves and mortality rate curves. The most commonly used survival curves are Kaplan-Meier survival plots, which illustrate the percent of live animals against time (Kaplan and Meier, 1958). Using these plots, the effects of specific experimental treatments on survival are analyzed by obtaining mean, median, and/or maximum survival times. Several statistical methods are employed for analyzing survival curves, such as the log-rank test and Fisher’s exact test (Fisher, 1990; Mantel, 1966). The log-rank test is used for comparing the survival distributions under two conditions and yields average survival times and
Mortality rate curves are drawn by calculating mortality rates, which are obtained by dividing changes in death incidence by time, and are used for deducing causes of deaths: accumulation of irreversible damage
In this review, we described survival assays using populations of