Q: The Arena instrument maintains internal temperature – why is this important for C. elegans studies?
A: The nematode C. elegans is commonly reared in the laboratory at temperatures between 15°C and 25°C, and worm growth and development drastically change even within this 10°C temperature range. Worms reared at 20°C and 25°C will grow 1.3 times faster and 2.5 times faster, respectively, than worms maintained at 16°C (Stiernagle 2006). Altering worm environmental temperature (either up or down) can result in altered gene expression, molecular interactions, individual behavior and ultimately population dynamics.
Simply considering gross phenotypes, a decrease in environmental temperature leads to an increase in C. elegans longevity. Maintaining worms at 20°C results in an average median lifespan of approximately 21 days; worms will grow approximately 10 days longer at 15°C and 10 days shorter at 25°C (Horikawa 2015). The laboratory “wild-type” C. elegans Bristol N2 strain also complies with the phenomenon known as the “temperature-size rule”, whereby ectotherm organisms mature at larger sizes under lower temperatures (Kammenga 2007). This is of course a prime consideration for the planning and execution of any assays involving median lifespan or body size as either experimentally directed variables or readout parameters. Inadvertent increase or decrease in analysis temperature can introduce unnecessary variability, confounding results and potentially distorting conclusions.
Finer phenotypes and behaviors such as pharyngeal pumping, egg laying, locomotion and chemotaxis are also extremely temperature dependent (whereas certain other behaviors, such as defecation, appear to be temperature independent). Pre-exposure of animals to higher temperatures initially results in increased activity, locomotion velocity and chemotaxis index (CI) as compared to controls; however, following an initial “normalcy” recovery period, these same animals then experience a deterioration of both locomotion and chemotaxis ability (Parida 2014). This indicates specific and quantifiable long-term physiological effects following even short-term exposure to changes in temperature.
Thermotolerance assays with C. elegans are most often performed at temperatures of 35°C or higher; depending on the age or larval stage of the worm, extended exposure to high temperatures can cause lethality in as little as two hours (Zevian 2014). Molecular-level physiological effects caused by high environmental temperatures include protein destabilization and degradation, halting of transcription and translation, and necrotic cell death (Park 2017). In order to most accurately assay genetic factors and pathways playing a role in thermotolerance, precise and consistent control of environmental temperature thus becomes absolutely necessary.
Parsing apart the genetic and molecular modulators of aging/longevity, healthspan and stress resistance likewise requires close attention to the environmental temperatures experienced by worms under analysis. In addition to the availability and utility of temperature-sensitive (ts) mutants, milder experimental phenotypes can exhibit full penetrance only under specific temperature conditions (Zhang 2015). Fine-tuning environmental temperature can therefore mean the difference between catching an interesting new mutant/observing a quantifiable result, and drawing an erroneous “no change” conclusion.
The NemaMetrix ARENA instrument, which contains an internal temperature control system that ranges from 20°C to 37°C, easily demonstrates the importance of consistent temperature maintenance during experimental analysis. As shown in Figure 1, change in analysis temperature from 20°C to 30°C results in a quantifiable difference in measured locomotion activity for each worm population. Similarly, as exhibited in Figure 2, a brief heat-shock of L3 larval stage worms as compared to Day 3 adult worms results in observable difference in measured locomotion recovery following return to the original rearing temperature of 23°C. Changes C. elegans’ environmental temperature can therefore significantly influence experimental results.
Figure 1: An increase in worm locomotion activity is observed with increasing temperature. Young adult Bristol N2 worms were plated and allowed to acclimate for 60min to the assay temperature; worm activity was then measured for 60min at the constant internal temperature shown using the ARENA instrument. Data obtained using 6-well NGM plates with no bacterial food source, containing 35 worms per well; data represents three technical replicates per temperature.
Figure 2: Recovery of L3 larval and Day 3 adult worms following 90min heat shock at 37°C. Bristol N2 worms of larval stage L3 and adult age Day 3 were reared at 23°C and analyzed for 30min in the ARENA instrument; a 90min heat shock at 37°C was introduced, following a recovery back to 23°C. As anticipated, adult Day 3 worms recovered back to pre-heat shock activity levels, whereas larval L3 stage worms were unable to recover to pre-heat shock activity levels. Data obtained using 6-well NGM plates with no bacterial food source, containing 35 worms per well; data represents three technical replicates per worm stage.
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