allowed to develop on one of three types of plates: NGM plates seeded with OP50, NGM plates without OP50, or aztreonam-LB/ NGM plates seeded with pre-azetreonam-treated OP50 and scored 15–20 hours later for spontaneous spicule protraction. The protocol for aztreonam-NGM plates and aztreonam treat- ment is given below. Males that crawled up on the side of the plate and dried up were not scored. To minimize suicidal males, we used an 8M glycerol ring around the edge of the plates. For daf- 2(e1368) lines, males were raised at 20uC until L2-L3 stage and then kept at 25uC for food-deprivation assays unless otherwise noted. For daf-2(m41) lines, males were raised at 15uC until L2-L3 stage and then raised and food-deprived at 25uC. For heat-shock rescue, males were heat-shocked at 33uC for 35 minutes prior to placing them on plates with or without bacteria. For all assays, males were picked from non-crowded and non-contaminated plates, and approximately 3–5 independent trials were performed. For mating efficiency assays, we separated L4 males from hermaphodites and placed them on one of the three different feeding conditions, similar to the above assay. The next day, we then placed one male with one pha-1(e2123) hermaphrodite on 3.5cm NGM plate that was seeded with a .5cm diameter lawn of log-phase E. coli OP50 15hrs prior to the assay. Males were allowed to mate for 2hrs and then removed from the plate. Plates were then incubated at 25uC for 48hrs and then scored for the presence or absence of pha-1(+) growing F1 progeny. pha-1(e2123) is a temperature-sensitive mutation that allows growth at 15uC, but is lethal at 25uC. Aztreonam-Treated OP50 (Inedible Food) OP50 cultures were grown to log growth in LB at 37uC with rigorous shaking. Once the cells reached log growth, aztreonam (Sigma) was added to a final concentration of 10 mg/ml, and the cells were grown an additional 3hrs with minimal shaking to minimize damage to the bacterial filaments. Cells were then spotted on LB plates containing NGM ingredients and 10 mg/mL aztreonam. Azteonam-treated bacteria and plates were used fresh (the same day), as plates older than 2 days had normal length bacteria, likely due to the drug being exhausted. Assay for the Effects of AWC Odorants on Spicule Protraction To phenocopy the effect of the AWC ablation and Podr-3:unc- 103(gf) experiments, we used odorants previously shown to reduce AWC activity. We used Sephadex G-50 beads (20–80 mm) (Sigma, www.sigmaaldrich.com) and acetone as a vehicle for the worms to chemotax to and contact. Specifically, we used a sterile M9 solution containing 1023 butanone, 1024 isoamyl alcohol, 1027 2,3-pentanedione, 1024 benzaldehyde, and 30mg/ml Sephadex beads. 20 ml of this solution was spotted on a sterile NGM plate along with 1 ml of .4M sodium acetate. As a control, we spotted 20 ml of a M9 solution containing only 30mg/ml Sephadex beads along with 1 ml .4M sodium acetate on a sterile NGM plate. The sodium acetate ensured that the worms remained near the soaked beads, and showed no effects on spicule protraction mutants when used in isolation. Visualization of Fat Stores using Nile Red Nile Red protocol was carried out as previously described [18]. Nile red was added to plates previously seeded with OP50, and larvae on these plates were synchronized by hypochlorite treatment. L4-stage males stained with Nile Red (www.mpbio. com) were then added to one of the three different feeding plates (OP50, aztreonam-treated OP50, or no OP50), then visualized for Sensory and Insulin-Like Signals Suppress Seizures Nile Red staining 15 hours later using a fluorescent microscope (100X). To quantify the number of fat droplets, equal planes and the region immediately posterior to the pharynx were selected. Hermaphrodites were also visualized and showed a similar trend; however the data shown in the figure were obtained from males. Analysis of Nile Red intensity changes were performed as described previously [18,85]. Briefly, images were captured by focusing on the first two intestinal cells. Nile Red intensity density was then quantified using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih. gov/ij/). A region of interest was drawn around the first two intestinal cells and a 1.0 pixel Gaussian filter was used to identify fluorescent lipid droplets. The Analyze Particles function in ImageJ was used to count the number of lipid droplets, and generate a mask. The mask was then overlayed to the original ROI marking the first two intestinal cells, resulting in measure- ment of fluorescence in only in lipid droplets. Intensity Density (area6mean) of fluorescence specifically in lipid droplets is reported in Figure 2D. Pharmacology Pharmacology was performed as previously described [1]. L4 males were separated from hermaphrodites and placed on either NGM plates seeded with OP50 or NGM plates with no OP50 for 15–20hrs, and then scored for drug sensitivity. Arecoline was obtained from Indofine Chemical Company (www.indofinechem- ical.com). Curve fits, EC90 and EC50 concentrations were generated using GraphPad Prism 4 software (GraphPad Software, Inc.). Plasmids The details for the generation of plasmids and primers used in this study are listed in the Supporting Information (Text S1 and Table S1). pTG81 contains pha-1(+) from pBX1 and the odr-3 promoter driving G-CaMP. pLR136 contains pha-1(+) and the unc-103E promoter driving G-CaMP. pTG71, pTG73, and pTG82 contain the ocr-2, odr-3, and osm-12 promoters driving unc-103(gf). pTG76, pTG78, and pLR89 contain YFP expressed from the odr-3, ocr-2, and osm-12 promoters, respectively. pTG57, pTG58, pTG59, pTG60, pTG61, and pTG65 contain the unc- 103E, aex-3, gtl-1, lev-11, tnt-4, and unc-103F promoters cloned in front of the daf-2 cDNA. pDG9 and pTG6 contain the lev-11 and tnt-4 promoters driving GFP and have been described previously [13]. pTG92 contains a 4.4kb upstream region of PLC-3 plus the first 12 codons fused to YFP. Transgenics To obtain unc-103 transgenic lines, DNA was co-injected with pBX1 (100ng/ml) into pha-1(e2131) unc-103(n1213) hermaphro- dites. The pBX1 plasmid contains pha-1(+) and was used to select for transformants [86]. For unc-103(gf) and YFP constructs, all constructs were injected at 25ng/ml. For the daf-2(+) rescue constructs, DNA was co-injected with a GFP-marker into daf-2(e1368) unc-103(n1213) hermaphrodites. The daf-2 rescue constructs and GFP markers were combined with pUC18 to bring the final DNA concentration to 200ng/ml and the injection mixtures for each rescue were: pTG60 (50ng/ml) and pDG9 (5ng/ml); pTG58 (50ng/ml) and pTG6 (20ng/ml); pTG57 (50ng/ml) and pDG9 (5ng/ml); pTG65 (50ng/ml) and pDG9 (5ng/ ml); pTG59 (50ng/ml) and pTG6 (20ng/ml); pTG61 (50ng/ml) and pTG6 (20ng/ml). The GFP-markers were used to select for transgenic males and at least two independent lines were analyzed for each injection. PLoS Genetics | www.plosgenetics.org 13 July 2008 | Volume 4 | Issue 7 | e1000117