(Punc-29:unc-29::YFP) provided a broader expression pattern than Punc-29:YFP. (A-D) Expression of Punc-29:YFP (yellow) and Pgar- 3:CFP (cyan) imaged from the tail of a same L4 larval male. The two images are merged to show that unc-29 is not expressed in the SPC neurons where gar-3 is expressing (C). The Nomarski image of the tail is also shown (D). Scale bar, 10 mm. (E, F) Expression of Punc-29:YFP in the male tail. Scale bar, 20 mm. We fused the 2.4 kb sequence upstream of the unc-63 start codon, including the first three codons, to the YFP gene. We found that this construct was expressed in the body wall muscles, the sexually dimorphic anal depressor muscle and sphincter muscle, and every male-specific sex muscle, including the spicule protractor and retractor muscles, the diagonal muscles, the gubernacular muscles, and the oblique muscles. However, it was also expressed in the SPC neurons where unc-29 was not expressed (G–J). When we made an unc-38 promoter-genomic DNA fusion construct, we found that, similarly to unc-63, it was also expressed in every male-specific muscle and the SPC neurons (K–N). (G–J) Expression of Punc-63:YFP in the male tail. Scale bar, 10 mm. (K–N) Expression of Punc-38:unc- 38::YFP in the male tail. Scale bar, 10 mm. (O, P) Expression of Pacr-12:acr-12::YFP in the male tail. Scale bar, 10 mm. (Q–S) Expression of Pacr-18:ChR2::YFP in the male tail. Scale bar, 10 mm. (T–V) Expression of Pacr-18:ChR2::YFP (yellow) and Ppkd- 2:CFP (cyan) imaged from the tail of a same adult male. The two images are merged to show that acr-18 is expressed in the HOA neuron that is next to the HOB neuron where pkd-2 is expressing (V). Scale bar, 10 mm. Found at: doi:10.1371/journal.pgen.1001326.s003 (2.78 MB TIF) Figure S4 Brief blue light pulses induce rapid repetitive spicule thrusts (method). Red boxes depict the region of interest (ROI) and are numbered. ROI 1 is used to detect the spicule displacement, and ROI 2 is to indicate the timing of blue light stimulation. (A) A representative frame taken when the blue light was off. The pixel intensity in ROI 2 was low. (B) A representative frame taken when the blue light was on. The pixel intensity in ROI 2 was high. (C) The standard deviation of the pixel intensity within ROI 1 (SDEV) was measured and plotted as a function of time (black trace). Changes in the SDEV indicate displacement of the spicule during and between the periods of light stimulation. The green trace presents the maximal pixel intensity in ROI 2 as a function of time. The increases indicate pulses of the blue light (see Materials and Methods). The grey regions also indicate the time periods of light stimulation. Panel a, b, c and d are representative frames of field 1 taken during and between blue light pulses, and changes in refraction of the ROI 1 region are indicated. The time points when these frames were taken are indicated in the ROI 1 SDEV trace (black). a.u. arbitrary units. Found at: doi:10.1371/journal.pgen.1001326.s004 (2.29 MB TIF) Figure S5 Brief blue light pulses induce rapid repetitive spicule thrusts (traces of individual males). Traces represent responses of individual males to blue light pulses. The method used is described in Figure S4. Black traces indicate displacement of the spicules during and between brief blue light pulses. The green traces indicate pulses of the blue light. The grey regions also indicate the time periods of light stimulation. (A–C) Three males that expressed active Pacr-18:ChR2::YFP, in the presence of all-trans retinal. (D–F) Three males that expressed inactive Pacr-18:ChR2::YFP, in the absence of all-trans retinal. Found at: doi:10.1371/journal.pgen.1001326.s005 (0.82 MB TIF) Figure S6 Correlation between the period of incubation on all- trans retinal and the efficiency of light-induced behavior. To minimize possible change in the G-CaMP/DeRed intensity ratio during incubation with all-trans retinal, we determined the minimal period of incubation that is sufficient to elicit ChR2-induced Schema of the Spicule Insertion Circuit behavior. We used males that expressed Pgar-3b:ChR2::YFP and males that expressed Pacr-18:ChR2::YFP. We allowed these males to be incubated on the all-trans retinal-containing plates for 0 min, 15 min, 30 min, 60 min and overnight. We then determined the percentage of the male population that protracted their spicules upon blue light stimulation for the Pgar-3b:ChR2::YFP males (upper panel), and determined the percentage of the male population that displayed rapid spicule thrusts upon light stimulation for males that expressed Pacr-18:ChR2::YFP (lower panel). We found in both cases, 30 min of incubation was sufficient to induce ChR2-elicited behavior and the efficiency was not significantly different from overnight incubation. The number of males assayed for each strain is listed within each bar (for 0 min, the number is above the X- axis). The number above the bar refers to the percentage of males displayed light-induced behavior. Asterisk (*) indicates the p value ,0.05, (**) indicate the p value ,0.001, (***) indicate the p value ,0.0001, using the Fisher’s exact test. Found at: doi:10.1371/journal.pgen.1001326.s006 (0.52 MB TIF) Figure S7 Carbenoxolone inhibition of blue light-induced repetitive spicule thrusts. CBX was injected at the concentration of 1 mM, 2.5 mM, 5 mM, 7.5 mM and 10 mM, to differentially suppress light-induced repetitive spicule thrusts in males that express Pacr-18:ChR2::YFP, Punc-103E:G-CaMP and Punc-103E:DsRed. ,10 males were assayed at each concentration. The X-axis plots concentration of the CBX, and the Y-axis plots the percentage of males that still displayed light-induced spicule thrusts. A log(agonist) vs. Normalized response-variable slope curve is fit to the data to estimate the minimal concentration of CBX that can inhibit light- induced spicule thrusts in the majority of the males. Found at: doi:10.1371/journal.pgen.1001326.s007 (0.14 MB TIF) Figure S8 Male tail expression of unc-9 and inx-14. Muscle abbreviations: DSP: dorsal spicule protractor; ADP: anal depres- sor; GER: gubernacular erector; GRT: gubernacular retractor; AOB: anterior oblique; POB: posterior oblique. (A-H) Expression of unc-9 in the adult male tail (B, D, F and H), and the corresponding DIC images of the male tail (A, C, E and G, respectively). (I-N) Expression of inx-14 in the adult male tail (J, L and N), and the corresponding DIC images of the male tail (I, K and M, respectively). All images were taken at 1006. Scale bar, 20 mm. The expression pattern was obtained by using transcriptional GFP fusion constructs (Altun et al. 2009) [55]. Found at: doi:10.1371/journal.pgen.1001326.s008 (2.05 MB TIF) Table S1 Primers used in this study. Found at: doi:10.1371/journal.pgen.1001326.s009 (0.03 DOC) MB Video S1 In the presence of all-trans retinal, brief blue light pulses induce corresponding rapid repetitive spicule thrusts in a male that expresses ChR2 in the gubernacular-oblique muscle group. Found at: doi:10.1371/journal.pgen.1001326.s010 (5.17 MB WMV) Video S2 In the absence of all-trans retinal, a male that expresses ChR2 in the gubernacular-oblique muscle group does not respond to blue light pulses. Found at: doi:10.1371/journal.pgen.1001326.s011 (4.16 MB WMV) Video S3 In the presence of all-trans retinal, Ca2+ transients are induced in the spicule protractors under blue light when ChR2 is expressed on the gubernacular-oblique muscle group. Found at: doi:10.1371/journal.pgen.1001326.s012 (0.76 MB WMV) PLoS Genetics | www.plosgenetics.org 18 March 2011 | Volume 7 | Issue 3 | e1001326