the hermaphrodites being paralyzed. When paired with free- moving hermaphrodites, the males could not maintain the contact (average duration = 2 seconds, n = 3). The contraction of the oblique muscles might change the tail posture during mating, based on their position and shape in the male tail. We propose that contractions of these muscles sustain vulval contact through the application of force between the male cloaca and the hermaph- rodite’s vulva. We then laser ablated the gubernacular erector and retractor muscles on both sides of the males at the early L4 larval stage, and observed their mating behavior when they became adults. The gubernaculum is a cuticle structure that lies posterior to the spicules, and it is thought to help guide the extension of the spicules through the male cloaca. The gubernacular erectors and retractors are muscles that attach the gubernaculum to the body wall [25,37]. We found that the vulval contact duration for these males was slightly, but not significantly shorter than the wild type (Figure 4C; Figure S2D); suggesting that unlike the oblique muscles, the gubernacular muscles are not essential to sustain vulval contact. Strikingly, the gubernacular muscle-ablated males showed difficulty in sperm transfer (Figure 4D; Figure S2E). In intact males, after sperm leaves the seminal vesicle, the gametes are briefly held at the distal end of the vas deferens for two to four seconds, until they are ‘‘released’’ out of the cloaca [2,20]. In gubernacular muscle-ablated animals, sperm accumulated at the distal end of the vas deferens and did not efficiently drain out of the male. In contrast, from casual observation, ablating oblique muscles or other spicule-associated neurons described in this work did not seem to affect this step of behavior. Sperm transfer requires signals from ventral cord neurons, the gonad and the SPV spicule sensory neurons [2,20,38]. We speculate that the gubernacular muscle contractions move the gubernaculum, which lifts the adjacent tissues so that the lumen of the distal vas deferens is accessible to the cloacal opening. The LEV-sensitive ACh receptor functions in the male sex muscles to facilitate vulval contact Previous data suggest that the p.c.s. neurons facilitate the males’ contact with the vulva through stimulating ionotropic ACh receptors on the postsynaptic oblique muscles. However, given the expression pattern of unc-29, it is also possible that the behavior is regulated by the L-AChRs on the ventral cord or head neurons. Therefore, we asked if restoring the L-AChR in the male sex muscles could reverse the vulval contact defect in the unc-29(lf);acr- 16(0) males. We restored unc-29 cDNA in the body wall muscles and the male sex muscles using the 3.5 kb promoter region of the tropomyosin gene lev-11 [6] (Plev-11:unc-29cDNA::SL2::GFP). Compared to unc-29(lf);acr-16(0) males that had unc-29 expressed only in the body wall muscles, males that expressed unc-29 in both body wall and sex muscles could sustain vulval contact significantly longer (Figure 4E; Figure S2F), suggesting that the L-AChR functions in the male sex muscles to facilitate vulval contact. The gubernacular-oblique muscle group activity directly induces repetitive protractor muscle contractions At the vulva, the male coordinates movements of multiple muscles in his tail to effectively penetrate the vulval slit. While he maintains his precise position at the vulva via the oblique muscles, his spicules are rapidly prodding the vulval slit as a result of repetitive protractor muscle contractions. The postcloacal sensilla initiate the spicule prodding behavior [22]; however, the postcloacal sensilla neurons do not make synapses to the protractor muscles (Figure 3B; Male Wiring Project). Schema of the Spicule Insertion Circuit The gubernacular erectors and retractors, as well as the anterior and posterior oblique muscles, are innervated by two of the three pairs of p.c.s. neurons or all three of them (Figure 3B), and it is likely that these muscles are stimulated simultaneously when the p.c.s. neurons become activated. In addition, the gubernacular retractors make gap junctions to both the anterior and the posterior oblique muscles (Male Wiring Project; Figure 3B). We therefore refer to these muscles as the ‘‘gubernacular-oblique muscle group’’. Interestingly, the spicule protractor muscles and the anal depressor muscle are also connected to the gubernacular erectors through gap junctions (Male Wiring Project; Figure 3B). Since gap junctions are low-resistance channels that bridge two cells and allow ions to pass directly, the cytoplasm of the cells are connected, and cell activity can be synchronized by exchanging electric transients [39,40]. We then hypothesized that ions or other signal molecules could pass from the gubernacular-oblique muscle group through the gap junctions to induce Ca2+ transients in the protractor and the anal depressor muscles. In this way, by innervating the gubernacular-oblique muscle group, the p.c.s. neurons could initiate repetitive contractions of the protractors. To test our hypothesis, we asked if localized activation of the gubernacular-oblique muscle group could immediately induce fast repetitive protractor muscle contractions and Ca2+ transients in these muscles. We employed the YFP-fused channelrhodopsin-2 light-gated cation channel (ChR2::YFP) to depolarize the gubernacular-oblique muscle group [41]. The 6.2 kb region upstream of the acr-18 gene was used as the promoter to drive transcription of ChR2::YFP in multiple cells, including the gubernacular erector and retractor muscles and the posterior oblique muscles, but not the spicule protractors (Figure S3Q and S3R). When ChR2 was expressed and activated by growing males in the presence of all-trans retinal, the activating blue light caused the spicule protractors to contract repetitively. When multiple sequential brief pulses of blue light were applied, the repetitive spicule movements were induced correspondingly, and coupled to the light stimulation (Figure 5A; Figure S4; Figure S5A-S5C; Video S1; see Materials and Methods). In contrast, males that grew in the absence of all-trans retinal did not respond to light stimulation (Figure 5A; Figure S5D-S5E; Video S2), since the blue light-induced transients require the presence of all-trans retinal [42]. To verify that the repetitive spicule movements were directly due to protractor muscle contractions and not to non-specific displacement of neighboring tissue, we measured Ca2+ dynamics in the spicule muscles. To detect Ca2+ level changes in the protractor muscles and the anal depressor muscle, we expressed a fluorescent Ca2+ indicator protein, G-CaMP, using one of the unc- 103 promoter regions (Punc-103E) [5,43,44], and expressed a red fluorescent protein (DsRed) using the same promoter as an internal control (Pacr-18:ChR2::YFP+Punc-103E:G-CaMP+Punc- 103E:DsRed) [5,45,46]. Ca2+ transients were determined by changes in the G-CaMP/DsRed intensity ratio in the same tissue. The excitation spectrum of G-CaMP overlaps with ChR2 [43,47]. To determine the baseline of the G-CaMP/DsRed intensity ratio in the protractor-anal depressor muscles (in the absence of ChR2- mediated gubernacular-oblique muscle activation), males were first imaged under the blue excitation light prior to exposure to all-trans retinal. Afterwards, they were incubated on plates that contained all-trans retinal for 30 minutes and then reimaged again under the blue light. 30 minutes of incubation was sufficient to activate ChR2 for light stimulation (Figure S6). In the presence of all-trans retinal, we observed Ca2+transients in correlation with light- induced contractions that caused muscle length changes (Figure 5B, 5C; Video S3; see Materials and Methods). In PLoS Genetics | www.plosgenetics.org 7 March 2011 | Volume 7 | Issue 3 | e1001326