recorded in the other field with a dimmer DIC image (field 2) (Figure S4A, S4B). Multiple blue light pulses were applied manually to each male tested. Once the blue light was turned on, the YFP fluorescent signal was detected in field 2 and this was used to indicate the timing of light stimulation in our recording (Figure S4C). An ROI was placed in the DIC image in field 1 at the base of one spicule (Figure S4A, S4B). During spicule thrusts, light refractivity of the region defined by the ROI changed, resulting in changes in the standard deviation of the pixel intensity within this ROI (SDEV) (Figure S4C). Therefore, the value of SDEV throughout the image sequence was used to indicate the displacement of spicule. Optical stimulation and Ca2+ imaging Strains used to image Ca2+ transient contained lite-1(ce314). Muscular Ca2+ transients were measured by detecting changes in fluorescence intensity of G-CaMP. The red fluorescent protein DsRed was expressed in the same set of cells as G-CaMP. Since the fluorescence intensity of DsRed does not change in response to light stimulation, it was used as an internal control. ChR2 is expressed in the gubernacular-oblique muscle group. DsRed and G-CaMP signals were recorded separately, but simultaneously via the Dual View Simultaneous Imaging Systems with an OI-11-EM filter by Photometrics (Surrey, BC). To record G-CaMP and DsRed, the transgenetic males were placed on agar pads without NaN3, and were observed under an Olympus BX51 microscope using a 406 objective. The images were recorded using a Hamamatsu ImagEM Electron multiplier (EM) CCD camera. Series of pictures were taken at the speed of ,25 frames per second for 1 minute after the blue light was turned on. To record Ca2+ changes, each transgenic male was separated from hermaphrodites at the mid-L4 larval stage, and grown on OP50 lawns overnight. The adult male was recorded for the first time with non-functional ChR2, to obtain the baseline ratio of G- CaMP/DsRed intensity (R0). This ratio does not change within a short period, since G-CaMP and DsRed expression were under control of the same promoter. Afterwards, they were incubated on OP50 lawns that contain all-trans retinal for 30 minutes and then reimaged under blue light. Laser stimulation and Ca2+ imaging Laser stimulation of muscles was slightly modified from a previous study [48]. 18–24 hr adult males containing G-CaMP and DsRed in their muscles were put on a 10% agarose (in H2O) pad containing 0.5 ml of 0.1 mm diameter polystyrene micro- spheres, and covered with a coverslip [87]. The minimum output of a Spectra-Physics VSL-337ND-S Nitrogen Laser was adjusted to induce muscle contraction. The laser was aimed at the gubernaculum erector or a posterior body wall muscle, and one to ten pulses were applied to induce the muscles to contract. G- CaMP and DsRed fluorescence changes were then measured in the gubernaculum and body wall muscles, as well as the anal depressor and protractor muscles. Analysis of Ca2+ imaging data The Hamamatsu SimplePCI (version 6.6.0.0.) software was used to analyze the movies. The region-of-interest (ROI) that covers the spicule protractor and anal depressor muscles was defined manually for both G-CaMP and DsRed, and an ROI was picked far away from the male as the background region for both channels. All ROIs had the same shape and area (by using the ‘‘ROI clone’’ tool), and the ‘‘mean grey’’ of the ROIs was calculated for each frame as the fluorescence intensity. The intensity of the background region ROI was subtracted from the Schema of the Spicule Insertion Circuit sample region ROI to exclude background fluorescence from other sources. The intensity of DsRed does not change in response to Ca2+ transients. The average intensity of the DsRed was calculated through the image sequence (DsRedaverage). The intensity of DsRed for each frame (DsRedn) deviates from the ‘‘DsRedaverage’’ only because of photo bleaching and/or changes in muscle shape, which should contribute to G-CaMP signal change as well. To cancel out these Ca2+-unrelated changes in G-CaMP signals, the intensity of G-CaMP in each frame (G-CaMPn) was then normalized by using the equation of ‘‘G-CaMPnormalized n = G- CaMPn 6 DsRedaverage/DsRedn’’. For the baseline recording before all-trans retinal incubation, a ratio of ‘‘G-CaMPnormalized n/DsRedaverage’’ for each frame was calculated (R0 n), and the average of this ratio within the first 2-4 seconds of recording (R0) was used as the baseline signal. For the second recording after all-trans retinal incubation, the ratio of ‘‘G-CaMPnormalized n/DsRedaverage’’ for each frame was also calculated (Rn). Finally, the Ca2+ level change (DR/R0) for this male was then calculated by ‘‘(Rn - R0)/R06100%’’. This ratio is comparable to the DF/F0 ratio used in other literature, except for that a G-CaMP/DsRed ratio is used instead of merely the G-CaMP intensity. The ratio DR/R0 was plotted over time as ‘‘corrected G-CaMP fold change trace for stimulated recording’’ (Figure 5B). The maximum fold change (max. DR/R0) throughout the frame sequence was then calculated. Finally, the ‘‘max. DR/R0’’ was used as the maximal Ca2+ level change for each male, and was plotted and compared between treatments groups using the Mann- Whitney non-parametric test (Figure 5C). Optical stimulation and continuous Ca2+ imaging A demonstration unit of the Mosaic Imaging System (Andor Technology) was used to image G-CaMP and DsRed in the protractor muscles before, during and after light-stimulation of the gubernacular-oblique muscle group. The image sequences were taken at a rate of ,72 frames/second. Males that expressed Pacr- 18:ChR2::YFP, Punc-103E:G-CaMP and Punc-103E:DsRed were used in this assay. Males were immobilized by using the 10% agarose pad and polystyrene microspheres described earlier. Only the region of protractors was illuminated and monitored in the first 100 frames of images (,1.4 seconds). Then, a region containing the gubernacular-oblique muscles was subsequently stimulated with blue light for 1000 frames of recording (,14 seconds). The protractors were continuously monitored during this period, and were recorded for another 500 frames (,6.9 seconds) after the end of gubernacular-oblique stimulation. The Ca2+ level change (DR/ R0) was then calculated by ‘‘(Rn - R0)/R06100%’’. Rn and R0 were determined using methods described earlier. For some males, periodic spicule movements were seen before stimulation of the gubernacular muscles, due to pressure applied on the male tail by the coverglass. RNA interference RNAi was induced by feeding worms bacteria producing double stranded RNA to the target genes. Bacteria containing the target genes were obtained from the C. elegans ORF-RNAi library [88]. Bacteria with the L4440 double-T7 vector but with no target gene was used as the negative control. Bacteria were grown and induced by IPTG using a standard protocol [89]. L4 males were transferred to plates spotted with the bacteria that express dsRNA and were incubated for ,20 hours. The adult males then were assayed for their response to light stimulation. The target gene PLoS Genetics | www.plosgenetics.org 16 March 2011 | Volume 7 | Issue 3 | e1001326