, 2009) ( Figure 2A). The locomotion rate and motile fraction

of pdf-1; npr-1 and pdfr-1; npr-1 double mutants during the L4/A lethargus were significantly lower than in npr-1 single mutants ( Figures 3D–3F). Inactivating PDF-1 and PDFR-1 had a much less dramatic effect on adult locomotion in pdf-1; npr-1 and pdfr-1; npr-1 double mutants ( Figure S3A). Thus, increased signaling by PDF-1 and PDFR-1 in npr-1 mutants was required for the increased motility during lethargus. The npr-1 foraging ZVADFMK defect was unaltered in pdf-1; npr-1 and pdfr-1; npr-1 double mutants ( Figure S3B), indicating that PDF was not required for other npr-1 phenotypes. Inactivating PDF-2 had little effect on the locomotion of npr-1 mutants during lethargus ( Figures S3C and S3D), indicating that PDF-1 is the major form of PDF involved in lethargus behavior. Collectively, these results suggest that PDF-1 functions as an arousal peptide in npr-1 mutants, preventing locomotion quiescence during lethargus. PDF-1’s effects on arousal were specific, because knockdown of BYL719 supplier 14 other neuropeptides expressed in the RMG circuit had no effect on the npr-1 lethargus defect ( Figure S3E). If PDF-1 functions as an arousal peptide, PDF-1 expression or secretion should be inhibited during lethargus, when animals are quiescent. We did several experiments to test this idea.

The abundance of pdf-1 and pdfr-1 mRNAs (assayed by quantitative PCR) was unaltered during the L4/A lethargus, whereas expression of mlt-10 (a gene required for molting) was significantly increased, as expected ( Figure S4A) ( Frand et al., 2005). To assay PDF-1 secretion, we expressed yellow-fluorescent-protein (YFP)-tagged proPDF-1 with the pdf-1 promoter ( Figures 4A and 4B). During DCV maturation, the YFP linked to proPDF-1

is cleaved by proprotein convertases and is subsequently secreted by DCV exocytosis. To assess the level of PDF-1 secretion, we analyzed PDF-1::YFP fluorescence in the endolysosomal compartment of coelomocytes, which are specialized scavenger cells that internalize proteins secreted into the body cavity ( Fares and Greenwald, 2001; Sieburth et al., 2007). either The PDF-1::YFP secretion reporter produced high levels of coelomocyte fluorescence in both L4 larvae and adults, whereas dramatically lower coelomocyte fluorescence was observed during the L4/A lethargus ( Figures 4A and 4B). Coelomocyte fluorescence produced by a second secretion probe (mCherry-tagged RIG-3 expressed in cholinergic neurons) ( Babu et al., 2011) was unaltered during lethargus ( Figure S4B), indicating that secretion and coelomocyte function were not globally inhibited during lethargus. If decreased PDF-1 secretion during lethargus is a cellular mechanism for inducing quiescence, we would expect that mutants retaining or lacking locomotion quiescence would exhibit reciprocal patterns of PDF-1 secretion during lethargus. We did several experiments to test this idea.