sequences in the bacteria were verified by PCR amplification and sequencing using the universal primers [88]. Injection of carbenoxolone and tubocurarine Males that expressed Pacr-18:ChR2::YFP, Punc-103E:G-CaMP and Punc-103E:DsRed were injected with the drugs. Carbenox- olone disodium salt (CBX) and (+)-Tubocurarine were purchased from Sigma-Aldrich (St. Louis, MO). Solution of CBX or tubocurarine was injected into the males using the same procedure as DNA microinjection. Males were imaged to obtain the baseline G-CaMP/DsRed ratio, and then were incubated on OP50 supplemented with all-trans retinal for 30 minutes. Drug or water was injected right after the incubation. These males were allowed to recover on OP50 supplemented with all-trans retinal for 1 hour, before they were imaged again to assess light-induced changes in the G-CaMP/DsRed ratio. For the injection needle not to stimulate any muscles or neurons in the male tail, one injection pulse was done at 1/3 of body length away from the posterior end of the male, the other was done in the anterior half of the male. A series of different concentrations of solution were injected for each drug to determine the proper concentration for our measurements. 7.5 mM or higher of CBX was found completely to suppress the light-induced repetitive spicule thrusts as well as the locomotion (Figure S7). Upon light stimulation, the G-CaMP signal then was measured in males that were injected with 7.5 mM CBX. The effects of different concentrations of tubocurarine were evaluated, and 7.5 mM or higher of tubocurarine was found to completely suppress the worm’s locomotion. 10 mM of tubocurarine then was injected for the recordings. Assay for blue light–induced behaviors All strains used in this assay contained the lite-1(ce314) allele. At L4, males expressing the respective transgenes were transferred onto NGM plates with OP50 supplemented with all-trans retinal, which was prepared in the same way as described. Males with the same transgenes were also placed onto NGM plates with regular OP50 as control. In the assay for light-induced sustained spicule protractor contraction, the SPC, PCA and PCB neurons were laser ablated in males at the mid-L4 larval stage. To remove hook associated cells, P10.p and P9.p were ablated at the L2 stage. The Plev- 11:ChR2::YFP expressing males were assayed for light-evoked behavior on 5% agar pads without NaN3. All other strains were assayed for light-evoked behavior on fresh all-trans retinal- supplemented OP50 plates, and their control males were assayed on standard OP50 plates. The behavior was observed using an Olympus SZX16 microscope and recorded using a Hamamatsu ImagEM Electron multiplier (EM) CCD camera. Males were illuminated with blue light from the EXFO X-Cite 120PC Q Fluorescence Illumination System, filtered with the SZX2-FGFPA GFP filter (Ex460-495/Em510-550). Males were filmed in the absence of blue light for the first few seconds and then blue light was manually turned on for ,10 seconds. Males that protracted their spicules in response to illumination for more than 5 seconds were counted as sustained spicule protraction positive. The unc-103(0) males occasionally protrude their spicules in the absence of mating stimulation. Eventually, ,30% of the virgin adult males that are separated from hermaphrodites will protract their spicules permanently. We used males whose spicules were not permanently protracted for the blue light stimulation assay. To rule out the possibility that spicule protraction scored was a result of spontaneous activity of the spicule circuit instead of light- stimulated activity, only males that retracted their spicules once Schema of the Spicule Insertion Circuit the light was turned off were counted as positive for light-induced spicule protraction. To determine the percentage of males that display rapid spicule thrusts upon blue light stimulation for the unc-9(lf) mutant and innexin RNAi strains, males were placed on 5% agar pads without NaN3, and observed under the Olympus BX51 microscope. The blue light was manually turned on for ,5 seconds and the males that displayed repetitive spicule thrusts during this period were counted as positive. Supporting Information Figure S1 Velocity of backward locomotion during vulval search behavior. The velocity of locomotion during vulval search behavior was measured for wild type and locomotion-restored unc-29(lf); acr-16(0) males. 5 males were measured for each strain. Average velocity for each strain is marked by the red bar, and the standard deviation is also shown for each group. No significant difference was found between the two groups (p = 0.15, t test). Found at: doi:10.1371/journal.pgen.1001326.s001 (0.19 MB TIF) Figure S2 The raw data of average duration of vulval contact for each male tested in Figure 1 and Figure 4. (A) Profiles of the average vulval contact duration (raw data for Figure 1B). The spots represent the average vulval contact duration of individual males. The horizontal bar indicates the sample median. Each mutant strain was compared to the wild-type males using the Mann-Whitney non-parametric test. (B–D) Raw data for Figure 4A-4C. The spots represent the average vulval contact duration for each individual male. The horizontal bar indicates the sample median. Ablated males are compared to control males that mated with the same group of hermaphrodites. Data that are shown in the same panel were obtained in parallel, but cannot be compared directly to data presented in different panels. (E) Ablation of the gubernacular muscles increases the duration of sperm release (raw data for Figure 4D). The spots represent the duration of sperm release for each individual male. The horizontal bar indicates the sample median. (F) Restoring unc-29 in male muscles reverses the vulval contact defect of the locomotion- restored unc-29(lf); acr-16(0) males (raw data for Figure 4E). The spots represent the average vulval contact duration of individual males. The horizontal bar indicates the sample median. Asterisks (**) indicate the p value ,0.005, (*) indicates the p value ,0.05, calculated using the Mann-Whitney non-parametric test. Found at: doi:10.1371/journal.pgen.1001326.s002 (0.61 MB TIF) Figure S3 Expression of ionotropic ACh receptor genes in the male tail. Muscle abbreviations: DSP: dorsal spicule protractor; VSP: ventral spicule protractor; DSR: dorsal spicule retractor; VSR: ventral spicule retractor; ADP: anal depressor; GER: gubernacular erector; GRT: gubernacular retractor; AOB: anterior oblique; POB: posterior oblique; SPH: sphincter. To obtain the expression pattern of unc-29 in the C. elegans male, we initially fused to the YFP gene a 2.2 kb sequence, which includes a 1.3 kb of sequence upstream of the unc-29 start codon and the genomic sequence of the gene up to the second intron (Punc- 29:YFP). This construct is expressed in the anal depressor muscle and the spicule protractor muscles in the male spicule circuit, and it is also expressed in the body wall muscles, some ventral cord neurons and some head neurons. This is consistent with the previous published expression pattern of unc-38, which encodes an a-subunit that forms the L-AChR with unc-29 (Liu et al. 2007). Nonetheless, the unc-29 genomic sequence also contains important information to facilitate transcription of the gene. Therefore, the expression construct that contained the unc-29 genomic sequence PLoS Genetics | www.plosgenetics.org 17 March 2011 | Volume 7 | Issue 3 | e1001326