Schema of the Spicule Insertion Circuit Figure 5. Stimulation of the gubernacular-oblique muscle group induces repetitive spicule thrusts and Ca2+ transients in the spicule protractor-anal depressor muscles. (A) Displacement of the spicule during and between brief blue light pulses. The grey regions indicate the time periods of blue light pulses. An image sequence of the male tail was captured for ,30 seconds when light pulses were applied repetitively. A region of interest (ROI) was placed at the base of a spicule, and the standard deviation of the pixel intensity within the ROI was obtained to indicate spicule displacement (see Materials and Methods). A representative trace for males that expressed active ChR2 (in the presence of all-trans retinal) is shown in the upper panel; a representative trace for males that expressed inactive ChR2 (in the absence of all-trans retinal) is shown in the lower panel. a.u. arbitrary units. (B) Raw fluorescence and corrected intensity traces for a representative male. Upper panel: raw fluorescence intensity traces in the protractor without gubernacular-oblique muscle stimulation for the baseline recording (with inactive ChR2), and during stimulation of active ChR2 on the gubernacular-oblique muscle group. Fluorescence intensity for both G-CaMP and DsRed channels are plotted, as well as the background fluorescence intensity for each channel. The periods that the light stimuli were applied are indicated by grey bars; the numbers on the X-axis indicate the time points since the onset of the most recent light stimulus; the 30 minute incubation is indicated on the X- axis. a.u. arbitrary units. Lower panel: corrected G-CaMP fold change trace during light stimulation with active ChR2. (C) Maximal G-CaMP intensity changes in the protractors during light stimulation. The number of males assayed for each operation is listed at the bottom of each bar. The numbers below the top of the bars indicate the mean of maximal G-CaMP intensity changes of the population. Error bars indicate the standard error of the mean. Asterisk (***) indicate the p value ,0.0005, (*) indicates the p value ,0.05, calculated using the Mann-Whitney non-parametric test. (D) Average G-CaMP intensity changes before, during and after a pulse of blue light stimulation. The blue arrow indicates the beginning of light stimulation, the red arrow indicates the onset of G-CaMP intensity increase, and the black arrow indicates the end of light stimulation. The trace in red represents males with active ChR2. Males without active ChR2 are represented by the blue trace. The grey region around each curve represents the standard error of the mean. 4 males were measured for each trace. (E) Averaged G-CaMP intensity change traces in the anal depressor muscle (ADP), the gubernacular erector (GER) and a posterior body wall muscle (BW), upon laser microbeam stimulation of the gubernacular erector muscle or the posterior body wall muscle in the male tail. Traces represent averaged G-CaMP intensity changes in specific muscles. The grey region around each trace represents the standard error of the mean. Left panel, the gubernacular erector on one side of the males was stimulated by a laser microbeam. The energy of the laser was adjusted to the lowest level that could elicit Ca2+ transients in the muscles. N = 10 males. Right panel, a body wall muscle that has no gap junction to the protractor-anal depressor muscle group was stimulated by a laser microbeam. Ca2+ transients in the gubernacular erector and the anal depressor muscles were detected by using G-CaMP. Stimulation of the posterior body wall muscle failed to induce Ca2+ transients in the anal depressor muscle, indicating that in the left panel, Ca2+ transients in the anal depressor muscle were not caused by non-specific laser damage in the male tail. N = 11 males. (F) Effects of innexin mutant, RNAi of innexins and SPC ablation on ChR2-induced spicule thrusts. The unc- 9(lf) males were compared to the wild type. The innexin RNAi strains with a wild-type genetic background were compared to the wild type that was fed with empty vector. The innexin RNAi strains with an unc-9(lf) genetic background were compared to the unc-9(lf) that was fed with empty vector- containing bacteria. The SPC-ablated wild type was compared to un-operated wild type. The numbers above the bars show the percentage of males that displayed rapid spicule thrusts upon light stimulation. The number of males assayed for each strain is listed within the bar. Asterisks (***) indicate the p value ,0.0001, using the Fisher’s exact test. doi:10.1371/journal.pgen.1001326.g005 contrast, ChR2-expressing males that grew in the absence of all- trans retinal, did not display spicule thrusts or had Ca2+ transients in the protractor muscles (data not shown, n = 30; Video S4). Pacr-18:ChR2::YFP was also expressed in the HOA hook sensillum neuron (Figure S3T-S3V), which theoretically could induce the spicule prodding behavior independent of the p.c.s. neurons [22]. To rule out the possibility that Ca2+ transients we observed in the protractor muscles were the result of HOA neuron activity, we laser ablated the hook sensillum precursor cell P10.p in the late L2 larval stage (see Materials and Methods). When we activated ChR2 in the gubernacular-oblique muscle group of ablated adult males, we found that Ca2+ transients in the protractor muscles were still induced with a magnitude that was slightly, but not significantly lower than mock-ablated males (p.0.05, Figure 5C). This result suggests that under these conditions, HOA is not the major contributor for Ca2+ transients observed in the spicule protractor muscles. To verify that Ca2+ transients observed in the protractor muscles were caused by excitation of the gubernacular-oblique muscle group, we ablated the gubernacular erector and retractor muscles and the anterior and posterior oblique muscles. We found that the repetitive contractions of the protractors were impaired, and Ca2+ transients observed in the ablated animals were greatly reduced (p,0.0005, Figure 5C). We reasoned that ablating nuclei at the mid-L4 larval stage severely impaired the physiology of these muscle cells and reduced the amount of ChR2 proteins on the remaining cell membranes. Thus, the cell corpses that remained after ablation could not be activated sufficiently to pass ions or signal molecules to the protractors to induce detectable Ca2+ transients. The remaining activity we saw in the protractors could be due to activity in the sphincter muscle and the spicule retractor muscles, since the upstream region of acr-18 also drove transcription in these muscles (Figure S3R, S3S). These muscles are also connected to the spicule protractors through gap junctions (Male Wiring Project). However in an intact male, due to the lack of innervation from male-specific neurons (Male Wiring Project), the sphincter and spicule retractor muscles are not likely to be the major facilitators of rhythmic spicule prodding behavior. These observations suggest that direct depolarization of the gubernacu- lar-oblique muscle group in the absence of neuronal input can elicit Ca2+ transients in the protractor-anal depressor muscles to induce muscle contraction. To gain a better idea of the temporal correlation between gubernacular-oblique muscle group depolarization and protractor- anal depressor muscle activities, we monitored Ca2+ levels continuously in the protractor muscles before, during and after a brief pulse of blue light stimulation of the gubernacular-oblique muscles. Using a Mosaic Imaging System (AndorTM Technology), we excited multiple regions simultaneously or sequentially without optically stimulating the whole field of view. We found that Ca2+ levels elevated gradually in the protractor muscles upon light- activation of the gubernacular-oblique muscles, and declined rapidly after the stimulation (Figure 5D; Video S5, S6; see Materials and Methods). We also stimulated the gubernacular erector muscles using a laser-induced muscle contraction method [48]. In males that did not express ChR2, abrupt release of internal Ca2+ stores in the gubernacular erector muscle, via irradiating the muscle with a low-energy laser microbeam (440 nm), also elicited Ca2+ transients in the protractor-anal depressor muscles, causing rapid muscle contraction (Figure 5E; Video S7, S8; see Materials and Methods). Interestingly, in both the optical and the laser stimulation of the gubernacular muscles, Ca2+ transients in the protractor and anal depressor muscles are induced with a delay of up to 0.3 seconds (Figure 5D; Figure 5E). The similar latency was also observed for light-induced spicule thrusts (Figure 5A; Figure S5). We speculate that this period is required for Ca2+ to travel between the muscles through gap junctions. Similar phenomenon was also observed in the C. elegans PLoS Genetics | www.plosgenetics.org 9 March 2011 | Volume 7 | Issue 3 | e1001326