Schema of the Spicule Insertion Circuit Figure 2. Male tail expression of acr-16 and unc-29. Gubernacular erector (GER), gubernacular retractor (GRT), anterior oblique (AOB), dorsal spicule protractor (DSP), ventral spicule protractor (VSP) and anal depressor (ADP) muscle. (A–D) Fluorescence images of the tail region of adult males expressing the Pacr-16:YFP construct. Scale bar, 20 mm. (E–H) Fluorescence images of the tail region of adult males expressing the Punc-29:unc-29::YFP construct. Scale bar, 20 mm. doi:10.1371/journal.pgen.1001326.g002 be expressed in the body wall muscles, the sexually dimorphic anal depressor muscle, and some male-specific muscles, such as the spicule protractor muscles, the gubernacular erector and retractor muscles and the anterior oblique muscles. However, it was not seen in any neuron in the spicule protraction circuit (Figure 2A- 2D). Similarly, a construct that has the 1.3 kb sequence upstream of the first exon of unc-29 and the whole unc-29 genomic sequence fused to the YFP gene was expressed in the same set of muscles as well as in some ventral cord neurons and some head neurons. This non-a L-AChR subunit was not expressed on any spicule neuron either (Figure 2E–2H; Figure S3A-S3F). UNC-63 and UNC-38 are a-subunits required for a functional L-AChR. We found both genes were expressed in the same set of male tail muscles as unc-29, but also expressed in the SPC spicule neurons (Figure S3G–S3N). This indicates that UNC-63 and UNC-38 probably form an ionotropic ACh receptor on the SPC neurons independent of UNC-29. UNC-63 and UNC-38 have been found to form a LEV-insensitive ionotropic ACh receptor with the a-subunit ACR-12 and also with the non-a-subunits ACR-2 and ACR-3 on the ventral cord cholinergic motor neurons [36]. This is consistent with the fact that the acr-12 promoter- genomic DNA fusion construct was also expressed in the SPC neurons (Figure S3O, S3P). Thus, by using the unc-29(e193) allele in the unc-29(e193);acr-16(ok789) double mutant, we only impaired the function of L-AChR on the male sex muscles. However, it was not initially obvious how reducing UNC-29 and ACR-16 in the sex muscles affected the male’s ability to maintain its position over the vulva during spicule insertion attempts. The postcloacal sensilla neurons maintain the male tail position at the vulva Electron micrograph reconstruction of the male has reveal extensive electrical and chemical communication between the bilateral sets of muscles and neurons in the tail region (Figure 3A) (S.W. Emmons, personal communication; S.W. Emmons, D.H. Hall, M. Xu, Y. Wang and T. Jarrell, Male Wiring Project, Albert Einstein College of Medicine, http://worms.aecom.yu.edu/pages/ male_wiring_project.htm). We studied the connections of the male nervous system to determine candidate neurons that make synapses to the acr-16 and unc-29 expressing male sex muscles. Among the muscles that express both acr-16 and unc-29, the spicule protractors are innervated by the SPC cholinergic neurons; the anterior oblique muscles (left/right), and the gubernacular erector (L/R) and retractor (L/R) muscles are innervated by the postcloacal sensilla (p.c.s.) neurons (Figure 3B). Previous work suggested that the precise location of the vulva is determined by three pairs of p.c.s. neurons, PCA, PCB and PCC. Operated males that contain any two of the three pairs of p.c.s. neurons can sense the precise position of the vulva [2]. However, males with zero or one pair of the p.c.s. neurons can sense the general area of the vulva via the hook sensillum neurons, but will pass over the precise location of the vulva without stopping [2,18]. Although these neurons had been shown to sense the vulva, it had not been determined if they also function in prolonging contact with the hermaphrodite’s genitalia. To assess these neurons’ role in maintaining vulval contact, we laser-ablated each individual pair of p.c.s. neurons and tested the male vulval contact efficiency. Similarly to published reports, we PLoS Genetics | www.plosgenetics.org 4 March 2011 | Volume 7 | Issue 3 | e1001326