To visualize PLC-3 expression, pTG92 (50ng/ml) was injected along with pBX1(100ng/ml) into pha-1 hermaphrodites. 8 independent transmitting lines were analyzed, one representative is shown in the Figure 5. Calcium Imaging with G-CaMP pLR136 (Punc-103E:G-CaMP) was injected at 12ng/ml and pLR135 (Punc-103E:ds-Red) at 2ng/ml into pha-1(e2123); lite- 1(e314) hermaphrodites (The lite-1(ce314) strain was graciously provided by Dr. Ken Miller, Oklahoma Medical Research Foundation). Ten independent transmitting lines were obtained and two lines with low to moderate background G-CaMP fluorescence were further analyzed. L4 males were separated from hermaphrodites the night before and allowed to mature into adults overnight. Worms were placed on a pad containing 2% Noble Agar dissolved in sterile water, using a mouth pipette filled with S-Medium. The agar slide containing the worm was then placed on a cold block (,1uC) for no longer than 15 seconds. This immobilized the male long enough to glue him down. A borosilicate capillary tube was pulled and broken, leaving an opening at the tip about .16mm in diameter. This tube was used to apply a small amount of glue (Nexaband S/C) to the male via mouth pipette near the dorsal posterior end of the animal. A drop of S-Medium was placed on top of the worm and then covered with a cover slip. Males were then observed using a 60x objective. The dorsal protractor muscles and anal depressor muscle were focused on and then the focus and exposure time were refined using fluorescence. Animals were then recorded for intensity changes using Image Pro Plus Version 6.2 software (Media Cybernetics). For drug exposure, a LED flash light was used to mark the beginning of drug application, and the drug was applied by pipetting 50 ml of drug the side of the coverslip. The drug then diffused across the pad and any intensity changes were recorded with Image Pro Plus. Fluorescence records were plotted as DF/F0, where F0 was the average baseline value of fluorescence before any drug stimulus. The sex muscles were marked as a region of interest using Image Pro Plus software, and the percent change in fluorescence intensity for this region was plotted for each time point relative to F0. To measure G-CaMP intensity changes in the AWC neurons, pTG81 was injected into pha-1(2123); lite-1(ce314) hermaphrodites at 35ng/ml. Six independent transmitting lines were obtained and one line with low background G-CaMP expression was used for analysis. Well-fed, virgin, adult males were washed with M9 buffer, and placed on a 1.5% agar pad containing no bacteria or sodium azide. Vacuum grease was then placed around the circumference of the pad followed by a cover-slip to seal in the moisture. Each male was labeled and intensity measurements were then recorded from the left AWC neuron at each time point of starvation at 100x. After starvation, males were removed from the agar plates, placed on NGM plates seeded with OP50 and allowed to re-feed for 15hrs. Males were then mounted again on the agar pads and the G-CaMP intensity in the left AWC neuron was recorded. At each time point, the fluorescence of the left AWC neuron and the background fluorescence of the isthmus of the pharynx were recorded. Intensity measurements were then plotted as the percent intensity change over time relative to the background fluorescence. RNAi of PLC-2 and PLC-3 To generate double-stranded RNA (dsRNA) for PLC-2, we PCR amplified a region of exon 9 using the primers FT7plc2 and RT7plc2 flanked by T7 promoter sequences. For PLC-3, we amplified exon 5 using the primers FT7plc3 and RT7plc3 flanked Sensory and Insulin-Like Signals Suppress Seizures by T7 promoter sequences. The resulting PCR products were then used a template for in vitro transcription with a T7-MEGAshort- script kit from Ambion (Austin, TX). The final RNA concentra- tion of each reaction was ,2 mg/ml. To perform RNAi, we placed L4 males in microfuge tubes containing 5 ml of dsRNA, and 15 ml of S-Medium. For controls with food, 2ul of concentrated OP50 was added as a food source along with 5 ml dsRNA and 13 ml S-medium. After 15hrs, males were removed from the microfuge tubes and scored for spontaneous protraction of their spicules. Laser Ablations Laser ablations were performed as described previously [87]. The odr-3 promoter driving YFP was used to identify the AWC neurons in unc-103(n1213) pha-1(e2123) males. Ablations were originally done at L4 stage, however no effect was seen, and the Podr-3:YFP construct still expressed in the AWC neurons even in the adult animal, suggesting that ablation in L4 males was too late. However, when ablations were done in L3 stage, YFP expression disappeared in the AWC neurons in adult males. To rule out any effects of the transgene or pha-1(e2123), ablations of AWC were then done in L3-stage unc-103(n1213) males and allowed to recover at 15uC overnight. The next day, males were moved to assay plates (food, inedible food, or no food) at late L4 larval stage and then scored for spontaneous spicule protraction 15–20hrs later. Males that dried up and died on the side of the plates were not scored. Supporting Information Figure S1 (A) Graph displaying chemotaxis index to isoamyl alcohol of males expressing chemosensory promoter-unc-103(gf) constructs. The * indicates a significant difference from non- transgenic controls (p-value , 0.05, Fisher’s Exact Test). (B) Representative image of a male that was fed non-treated GFP- expressing E. coli. Arrow points to intact E. coli in the intestines (C) Representative image of a male fed aztreonam-treated GFP- expressing E. coli. Arrow points to inedible-aztreonam treated E. coli. (D) Graph displaying the number of cell nuclei observed in the gonad of L1-stage worms placed on one of the three feeding conditions for 15hrs (p-value Fisher’s exact test). (E) Graph displaying the effect of Podr-3:unc-103(gf) and AWC ablation on wild-type male mating efficiency (p-value Fisher’s exact test). Found at: doi:10.1371/journal.pgen.1000117.s001 (3.93 MB TIF) Figure S2 (A) Representative pictures of males expressing LGG- 1 in 3 separate feeding conditions, Fed, Food Deprived for 15hrs, and Food Deprived for 3 days (scale bar 9 mm). (B) Graph displaying the number of LGG-1 puncta in males under four different conditions, Fed, Inedible Food 15hrs, No Food 15hrs, and starved for 3-days. For each condition, 10–20 males were analyzed. The * indicates a significant difference (p,0.001) then the other 3 feeding conditions. (C) Representative images of fed and food-deprived males stained with phalloidin, which stains filamentous actin (scale bar 20 mm). No differences were observed in muscle structure between the two conditions. Found at: doi:10.1371/journal.pgen.1000117.s002 (3.30 MB TIF) Table S1 Primers used in this study. Found at: doi:10.1371/journal.pgen.1000117.s003 (0.04 DOC) Text S1 Supporting materials and methods. Found at: doi:10.1371/journal.pgen.1000117.s004 (0.06 DOC) MB MB PLoS Genetics | www.plosgenetics.org 14 July 2008 | Volume 4 | Issue 7 | e1000117