2 Neuroscience Department, Oberlin College, Ohio
3 Neuroscience Program, University of Illinois Urbana-Champaign
These findings suggest that IIS is reduced comparably at 15°C and 25°C and argues against the assumption that e1370 is necessarily a temperature-sensitive allele in the classic sense where temperature affects protein folding and activity. Interestingly, about 10% wild-type worms form dauers when grown at 27°C under otherwise standard culturing conditions (ample food and not overcrowded; (Ailion & Thomas 2000 PMID:11063684)). Furthermore, it has been observed that null mutants in other abnormal-DAuer-Formation (daf) genes than daf-2, only show the dauer phenotype at higher temperatures, suggesting that the dauer phenotype itself is temperature sensitive. This observation was published over 30 years ago (Golden & Riddle 1984) and is discussed in (Wood 1988).
We are motivated to write to the WBG community by the assumption that during adulthood daf-2(e1370) mutants improperly reactive phenotypes that are reminiscent of dauer larvae (reduced mobility, reduced brood-size, short body size; (Gems et al. 1998)). Recently, we have shown that daf-2(e1370) mutants form dauers independent of SKN-1/NRF1,2,3 and that SKN-1 is only required for the daf-2(e1370)-longevity phenotype when these dauer-related phenotypes are not activated during adulthood (Ewald et al. 2015). Furthermore, comparing expression profiles of daf-2(e1370) mutants with dauer larvae revealed that they share only a common overlap under conditions that reactivate these dauer phenotypes (e.g., daf-2(e1370) at higher but not at lower temperatures; (Ruzanov et al. 2007 PMID:17543485; McElwee et al. 2004 PMID:15308663; Ewald et al. 2015)). Finally, treating wild-type worms with dauer pheromone was sufficient to increase lifespan (Kawano et al. 2005 PMID:16377915; Ewald et al. 2015). This suggests that there might be a dauer program activated in daf-2(e1370) mutants at higher temperatures that masks other underlying mechanisms of reduced IIS on lifespan. Further research is required, however, these findings suggest that reduced IIS can extend lifespan via dauer-dependent and dauer-independent mechanisms and this changes the interpretation of experiments performed with daf-2(e1370) mutants under conditions that promote these dauer-associated phenotypes.
Golden, J.W. & Riddle, D.L., 1984. A pheromone-induced developmental switch in Caenorhabditis elegans: Temperature-sensitive mutants reveal a wild-type temperature-dependent process. Proceedings of the National Academy of Sciences of the United States of America, 81(3), pp.819–823.
Wood, W.B. ed., 1988. The Nematode Caenorhabditis elegans. 1st ed., Cold Spring Harbor Monograph Series.
During larval development C. elegans has two distinct time points where it can arrest development until environmental conditions become more favorable. The first one is L1 arrest, which occurs when worms hatch in the absence of food. The second decision point is later, around L1-L2 molt, and if conditions are poor at this time (little food, crowding, and high temperature) worms gain the potential to become dauers. L1 arrest and dauer formation are not mutually exclusive and, in principle, a worm can go through both of them sequentially. However, it is not known whether L1 arrest and dauer formation are coupled, i.e., if experiencing L1 arrest affects a worm’s decision to become a dauer. A naïve prediction would be that worms that experienced L1 starvation should be more susceptible to become dauers since they were already exposed to a harsh environment.
We studied dauer formation of N2 worms in liquid and found that in reality worms that experienced L1 arrest and starvation were less likely to become dauers. In a typical experiment, eggs obtained from an egg prep were incubated at 20 0C in S-complete with a small amount of bacterial food (HB101) and dauer formation was assessed 6 days later based on survival in 1% SDS (Pungaliya et al., 2009). When we let eggs hatch without food and added it 1-4 days later, keeping other conditions the same, significantly fewer worms became dauers (Figure 1). This was not due to slower development after starvation and incomplete dauer formation. We also found that this effect reflected an internal state of the worm rather than exposure to L1 exometabolome during L1 arrest since worms starved as L1s at low and high density later formed dauers with the same probability (Figure 1, during the dauer formation worms were at the same low density of 1 worm/µl to minimize the effect of released chemicals, see below).
Starved L1 worms release numerous metabolites, including several ascarosides, and we anticipated that they might promote dauer formation. Indeed, when S-complete was substituted with conditioned medium from high-density L1 larvae starved for 24 h, more worms became dauers (Fig. 2), consistent with the known dauer-inducing effect of crowding mediated by ascarosides (Ludewig and Schroeder, 2013). However, this result apparently contradicts the starvation effect described above. On one hand, when a worm itself experienced L1 arrest but received no signal from other worms (low density condition), it was less likely to become a dauer and preferred to try to grow to adult. On the other hand, a worm that had not experienced starvation itself but was exposed to chemical signals released by other starved L1 worms, was more likely to become a dauer. The next obvious experiment is to combine these two opposite effects and see which one is stronger. The result of this test (Fig. 2) is that the external starvation signal (mediated by L1 conditioned medium) overrides the internal one (L1 arrest). When making a decision whether to become a dauer, apparently C. elegans worms trust the communal voice more than their own.
While the dauer-inducing effect of L1-conditioned medium is intuitively easy to accept, the negative effect of L1 starvation on subsequent dauer formation is less clear. A recently published model of contrast effects (McNamara et al., 2013) may provide a theoretical framework for understanding this phenomenon. But to keep us from being too comfortable with our simplistic views nature presented us with another puzzle: L1 arrest does not negatively affect dauer formation in C. briggsae.
Pungaliya C, Srinivasan J, Fox BW, Malik RU, Ludewig AH, Sternberg PW, and Schroeder FC. (2009). A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans. Proc. Natl Acad. Sci. U. S. A. 106, 7708-7713.