Working with starved L1 larvae of C. elegans and C. briggsae we noticed that these two species behave quite differently in starvation. First, C. elegans adults stop laying eggs after exhausting bacterial food, which eventually leads to internal hatching and bagging. C. briggsae do not show this behavior. This difference has been observed before (McCulloch and Gems, 2003). Second, at high enough density of worms, arrested C. elegans L1s aggregate on agar plates after several days of starvation (Fig. 1a). C. briggsae L1s do not form aggregates (Fig. 1b). Aggregation may serve several purposes ranging from decrease of surface to volume ratio and use of diffusible “public goods” to sharing information about quality of the environment. Third, survival of starved C. elegans L1s strongly depends on their density – the higher the worm density, the longer they survive (Fig. 1c) (Artyukhin et al., 2013). This holds true for starvation on plates as well as in suspension. Survival of C. briggsae L1s is independent of worm density (Fig. 1d). We believe that the aggregation and the density dependence are naturally connected as they seem to be correlated across Caenorhabditis species. Forth, starved C. elegans L1s release plenty of ascarosides (Fig. 1e,f). C. briggsae L1s release far less, both in variety (Fig. 1e) and in amounts (Fig. 1f). More than 100 ascarosides have been found in C. elegans (von Reuss et al., 2012), and although we do not know physiological functions for many of them, functions of the ones that we do know and the high degree of structural conservation in other nematodes suggest that ascarosides constitute a vital part of nematode chemical language. Hence, we interpret the data in Fig. 1e,f as suggesting that C. elegans L1s “talk” more with each other in starvation than C. briggsae. Finally, C. elegans is one of a couple Caenorhabditis species susceptible to environmental RNAi (Winston et al., 2007), which has been speculated to play a role in communication between organisms (perhaps, conspecifics) (Whangbo and Hunter, 2008). Based on everything above, we speculate that in unfavorable conditions (starvation) C. elegans tend to become more social than C. briggsae. In variable and unpredictable environments genotypic fitness can be maximized either by reducing individual-level variance in fitness or by reducing between-individual correlations in fitness (or some combination of the two) (Starrfelt and Kokko, 2012). Aggregating or social species may choose to minimize individual-level variance by having a mechanism that helps them to adjust to unfavorable conditions, in part through collective behavior. Non-aggregating species may use dispersal as a strategy to minimize between-individual correlations. Based on this hypothesis we would predict that lack of aggregation and density dependence in C. briggsae implies that their starved L1s hardly ever find themselves at high density in nature and the optimal strategy for them is to disperse and actively look for food.
Like humans, C. elegans lacks uricase, an enzyme responsible for oxidation of uric acid to allantoin (Ramazzina et al., 2006). Thus one would expect uric acid to be the final product of the purine degradation pathway. The presence of allantoin can be rationalized in two ways. First, uric acid may be oxidized to allantoin by some other enzyme, e.g., a cytochrome P450. In humans, myeloperoxidase has been shown to catalyze this step (Meotti et al., 2011). Second, uric acid is a known antioxidant (Ames et al., 1981), and its oxidation can proceed nonenzymatically, e.g., in the presence of ROS, with allantoin being one of the oxidation products.
We profiled mRNA levels in arrested L1 larvae from plate- and liquid culture-grown mothers. We found 145 genes that were different between these two groups by a factor of 3 or more (http://elegans.som.vcu.edu/~alex/purine_metabolism). Half of them were genes encoding proteins with unknown function or homology. sod-3 and sod-5 were overexpressed in L1 progeny from plates compared to liquid culture (3 and 4.1 fold difference). This might indicate that progeny from plates have better ROS protection, while liquid culture progeny are more susceptible to oxidative stress and uric acid acts there as an endogenous antioxidant, resulting in its nonenzymatic oxidation to allantoin. We then compared uric acid and allantoin levels in L1 medium of sod-3(tm760), sod-5(tm1146) and N2 whose parents were grown on plates and found no significant difference. We also measured uric acid and allantoin concentrations in L1 medium (both from plates and liquid culture) in the presence of 200 µM epicatechin, an antioxidant that was shown to reduce oxidative stress in adult C. elegans (González-Manzano et al., 2012). Epicatechin had no effect on uric acid and allantoin levels. Overall, these experiments suggest that different levels of oxidative stress may not explain differences in the uric acid/allantoin ratio.
The microarray data showed that the P450 family gene cyp-13A5 is upregulated 5.4 fold in liquid culture progeny, so we hypothesized that cyp-13A5 might catalyze oxidation of uric acid to allantoin. RNAi knockdown of cyp-13A5 in liquid culture-grown mothers did not cause a change in uric acid and allantoin levels in L1 medium of the progeny. In the same conditions unc-22 RNAi caused a partial uncoordinated phenotype, but we did not verify cyp-13A5 knockdown level by qPCR, so this result may not be conclusive.
In conclusion, we found that in arrested L1 worms whose parents were grown on plates purine metabolism stops at uric acid, while in arrested L1s whose parents were grown in liquid culture it proceeds one step further and uric acid is converted to allantoin. At present, we do not have a satisfactory explanation for this effect. Finally, we found that there was no significant difference in L1 starvation survival between these two groups of worms.
We thank Maciej Kukula for the help with HPLC-MS.
Ames BN, Cathcart R, Schwiers E, Hochstein P. (1981) Uric acid provides an antioxidant defense in humans against oxidant- and radical-caused aging and cancer: a hypothesis. Proc Natl Acad Sci U S A 78, 6858-6862.
González-Manzano S, González-Paramás AM, Delgado L, Patianna S, Surco-Laos F, Dueñas M, Santos-Buelga C. (2012) Oxidative status of stressed Caenorhabditis elegans treated with epicatechin. J Agric Food Chem 60, 8911-8916.
Johnson RJ, Sautin YY, Oliver WJ, Roncal C, Mu W, Sanchez-Lozada LG, Rodriguez-Iturbe B, Nakagawa T, Benner SA. (2009) Lessons from comparative physiology: could uric acid represent a physiologic alarm signal gone awry in western society? J Comp Physiol B 179, 67–76.
Meotti FC, Jameson GNL, Turner R, Harwood DT, Stockwell S, Rees MD, Thomas SR, Kettle AJ. (2011) Urate as a physiological substrate for myeloperoxidase: implications for hyperuricemia and inflammation. J Biol Chem 286, 12901-12911.
Survival of L1 arrested larvae in the absence of food is a common experiment in C elegans lifespan studies. A typical protocol involves resuspension of freshly bleached worm eggs in sterile inorganic buffer. Newly hatched worms arrest due to lack of food and their starvation survival in the following days is monitored by taking aliquots and counting live worms. It is tempting to think that such well defined and simple experimental conditions leave little room for variability. In reality, the outcome of this experiment is sensitive to a number of often neglected factors ranging from worm maintenance history dating several generations back to small temperature fluctuations during starvation. Recently, we found additional sources of artifacts in L1 survival assays.
When starved L1 larvae are incubated in M9 buffer in plastic tubes (e.g. polypropylene 15-ml tubes), worms tend to adhere to hydrophobic plastic walls, which decreases apparent worm counts in suspension. This effect becomes particularly noticeable at low worm densities (< 3 worms/μl, Fig. 1) We found that precoating tubes with bovine serum albumin (1% aqueous solution for an hour followed by 3 water rinses) renders plastic hydrophilic and greatly reduces the sticking problem. BSA solution and water for rinses should be sterile.
When the starvation experiment is performed in glass tubes or vials, worm sticking is not a problem, since clean glass is hydrophilic. The danger in this case comes from the cap, since seemingly inert material lining the cap can significantly affect survival rate if it comes in contact with liquid. We found that L1 worms starved in glass tubes with rubber-lined caps reproducibly survived starvation longer then worms in plastic tubes of the same volume (Fig. 2) or glass tubes with teflon-lined caps (Fig. 3). We detected several compounds in water from tubes with rubber-lined caps and identified one of them as 2-mercaptobenzothiazole (2-MBT), which was present at ca. 0.5 μM. 2-MBT, used as an accelerator in the vulcanization of rubber, is commonly seen in rubber leachates (Reepmeyer and Juhl, 1983). We speculate that longer survival is due to a hormetic effect of one or several chemicals leaching from rubber, which would be toxic at higher concentrations. However, low concentrations of synthetic 2-MBT alone were not sufficient to reproducibly extend L1 starvation survival (at 3 μM and above 2-MBT is toxic to worms). Other components of the rubber leachate may be necessary for the effect.
These results demonstrate artifacts that can both decrease and increase apparent survival rates in L1 starvation.