Schema of the Spicule Insertion Circuit Figure 4. The postcloacal sensilla (p.c.s.) neurons and the oblique muscles facilitate prolonged vulval contact. (A–C) The average duration of vulval contact for each male was normalized to the mock-ablated mean to obtain the ‘‘normalized score for vulval contact duration’’ (Materials and Methods). The mean of normalized score for each strain is represented by the height of the bar, and it is also indicated by the number below the top of the bar. Error bars indicate the standard error of the mean. The number of males assayed for each operation is listed within each bar. Ablated males are compared to mock-ablated 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. (D) Ablation of the gubernacular muscles increases the duration of sperm release. The average duration of sperm release is represented by the height of the bar, and it is also indicated by the number below the top of the bar. The number of males assayed for each operation is listed within each bar. Error bars indicate the standard error of the mean. (E) Restoring unc-29 in male muscles reverses the vulval contact defect of the locomotion-restored unc-29(lf);acr-16(0) males. The average duration of vulval contact for each male was normalized to the mean of the body wall muscle unc-29 rescued males, to obtain the ‘‘normalized score for vulval contact duration’’. The mean of normalized score for each strain is represented by the height of the bar, and it is also indicated by the number below the top of the bar. The number of males assayed for each operation is listed within each bar. Error bars indicate the standard error of the mean. Asterisks (**) indicate the p value ,0.005, (*) indicates the p value ,0.05, calculated using the Mann-Whitney non-parametric test. The raw values of average duration of vulval contact or duration of sperm release for each male tested are presented in Figure S2B-S2F. doi:10.1371/journal.pgen.1001326.g004 [22]. The non-cholinergic PCA neurons might exercise this function using other neurotransmitters. The cholinergic SPC neurons innervate the spicule protractor muscles and are required for sustained protractor contraction, which results in keeping the spicules inserted into the hermaph- rodite’s vulva during sperm transfer [22,25]. We observed the effect of ablating the SPC neurons on vulval contact duration, and found that unlike the p.c.s. operations, the SPC-ablated males could stay at the vulva of paralyzed hermaphrodites significantly longer than the intact control males (Figure 4B; Figure S2C). This increase in duration is not a simple result of the failure to insert the spicules, since hermaphrodites used for this assay were purposely chosen to be difficult for intact males to penetrate. Additionally, intact control males that were observed to penetrate the hermaphrodite, inserted their spicules after multiple vulval contacts, and multiple contacts were recorded for the SPC operated males as well (see Material and methods). In addition, ablating the SPC neurons could reverse the vulval contact deficiency caused by ablation of an individual pair of p.c.s. neurons (PCCs) (Figure 4A; Figure S2B). These indicate that, unlike the p.c.s. neurons, the SPC neurons are not required to sustain contact with the vulva, but might be involved in delimiting the duration of this step in mating behavior. The oblique muscles are required for prolonging vulval contact To test the hypothesis that the male p.c.s. neurons maintain vulval contact by innervating the acr-16 and unc-29 expressing sex muscles, we ablated the oblique muscles and the gubernacular muscles, to see which operation causes defects similarly to the p.c.s. ablations. We ablated both anterior and posterior oblique muscle nuclei in L4 males. The anterior and posterior oblique muscles lie dorsal-ventrally at the male tail near the cloacal opening, having both the dorsal and ventral ends attached to the body wall [25,37]. Similarly to intact males, the operated males initiated backward scanning of the hermaphrodites, and they paused at the vulva and started to prod the vulva with their spicules. However, they lose vulval contact and resumed backward scanning more frequently than non-ablated males (Figure 4C, Figure S2D). The vulval contact that remained was largely due to PLoS Genetics | www.plosgenetics.org 6 March 2011 | Volume 7 | Issue 3 | e1001326