The duration of pharyngeal pumping quiescence was unaltered in npr-1 mutants, AUY922 indicating that the duration of lethargus had not been altered ( Figure 1A). Pharyngeal pumping rate was also unaltered in npr-1 adults ( Figure 1B). To assess changes in locomotion during the L4/A lethargus, we analyzed the fraction of time

animals undergo active motility (motile fraction) and locomotion velocity. Unlike wild-type animals, npr-1 mutants exhibited fast and nearly continuous locomotion during the L4/A lethargus ( Figures 1C–1E; Movies S1 and S2 available online). The effects of npr-1 on locomotion persisted throughout the L4/A lethargus (as defined by pumping quiescence) ( Figures S1A and S1B). Inactivation of npr-1 had a significantly larger effect on locomotion during the L4/A lethargus Autophagy screening (motile fraction, 17-fold increase; velocity, 50-fold increase) than in adults (motile fraction, 1.2-fold increase; velocity, 2-fold increase) ( Figures S1C and S1D). These results suggest that NPR-1 is required for locomotion quiescence during lethargus, but not for feeding quiescence.

The npr-1 gene is polymorphic among wild-type populations, with two frequent alleles observed (215V and 215F) ( McGrath et al., 2009; Weber et al., 2010). These wild-type alleles encode receptors that differ in their affinity for NPR-1 ligands (FLP-18 and FLP-21), with 215V exhibiting higher affinity (and lower half-maximal effective concentration values) than 215F receptors ( Kubiak et al., 2003; Rogers et al., 2003). To determine whether wild-type strains are also polymorphic for lethargus behavior, we analyzed locomotion during the L4/A lethargus ( Figures 1D and 1E). All 215V-containing strains exhibited similar levels of quiescence and were significantly more quiescent than 215F

strains. The quiescence observed in 215F strains was more variable, with one strain (RC301) exhibiting L4/A locomotion similar to that of npr-1 null mutants and other strains Liothyronine Sodium (AB3 and CB4856) exhibiting intermediate levels of quiescence. Thus, the extent of behavioral quiescence during lethargus is polymorphic among wild-type strains. A strain carrying a 215F allele (g320) in the Bristol genetic background had significantly stronger quiescence than was observed in unrelated 215F wild-type strains (e.g., CB4856 and RC301). These results suggest that variation in genes other than npr-1 also contribute to differences in the lethargus behaviors of wild-type strains. Two NPR-1 ligands have been identified, the neuropeptides FLP-18 and FLP-21 (Kubiak et al., 2003; Rogers et al., 2003). Both neuropeptides bind and activate NPR-1 receptors expressed in transfected cells; however, NPR-1 exhibits significantly stronger affinity for FLP-21.