Figure 1. Ionotropic ACh receptors are required for sustained vulval contact. (A) Relative mating potency of mutant males normalized to wild type. The number of males assayed for each strain is listed within each bar. The number above the bar refers to the normalized mating potency value. The males were mated with free- moving hermaphrodites. Each mutant strain was compared to the wild- type males that were assayed in parallel, using the Fisher’s exact test. Asterisks (***) indicate the p value ,0.0001. (B) Profiles of the vulval contact duration. The males were mated into paralyzed hermaphro- dites. The average duration of vulval contact for each male was normalized to the wild type mean to obtain the ‘‘normalized score for vulval contact duration’’, as a measurement of the male’s ability to maintain precise vulval contact (Materials and Methods). The mean of the normalized score for vulval contact duration for each strain is Schema of the Spicule Insertion Circuit 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 strain is listed within each bar. Each mutant strain was compared to wild-type males using the Mann-Whitney non-parametric test. Asterisk (*) indicates the p value ,0.005. The raw values of average duration of vulval contact for each male tested are presented in Figure S2A. doi:10.1371/journal.pgen.1001326.g001 male mating behavior, and unlike the locomotion behavior, if the function of one receptor is compromised, the other can equally compensate. We then asked if the locomotion-restored double mutant males mated poorly because they could not insert their spicules, or if they were additionally defective in other steps of mating, such as maintaining contact with the hermaphrodite’s vulva. We paired males with paralyzed unc-64(lf) hermaphrodites for them to mate, so that duration of each vulval contact could be accurately recorded [26]. During a 5 minute observation window, the majority of the males we assayed made multiple contacts with the hermaphrodites’ vulva before they inserted their spicules or until the observation period ended. Then we determined the average duration of the vulval contacts for each male (Figure S2A). In order to score his ability to maintain precise vulval contact, we first normalized wild type to a score of 1 and then we compared the normalized mutant males to the wild type (Materials and Methods) (Figure 1B). We found that the unc-29(lf);acr-16(0) males sustained vulval contact for half the time of wild-type males (p,.05, Figure 1B; see Materials and Methods). In contrast to matings with moving hermaphrodites, the reduced vulval contact did not severely affect the mating potency of the mutant males when they mated with paralyzed hermaphrodites. 48.5% of the double mutant males inserted their spicules and transferred sperm into unc-64(lf) hermaphrodites during the 5 minute observation period (n = 33), not statistically different from the 69.7% of the wild type (n = 33, p = 0.13). However, failure to sustain their position over the vulva is likely to be the reason why the double mutant males cannot inseminate free-behaving hermaphrodites, as maintaining contact with a moving object is already challenging for wild-type males: only 10% of the wild-type males could inseminate free- moving hermaphrodites within 5 minutes (n = 20). We then asked if the defect in maintaining vulval contact in unc-29(lf);acr-16(0) males was caused by the loss of the L-AChR or the ACR-16 nAChR, or both. Loss of function of either receptor did not reduce the vulval contact duration with statistical significance (Figure 1B), suggesting that these genes are used redundantly to maintain vulval contact. However, although the average duration of vulval contact were not statistically significant between wild type and the mutants, the distributions of vulval contact duration were slightly different (p,0.005, F test; Figure S2A) between wild type and the unc-29(lf), as well as between wild type and the acr-16(0) mutant, suggesting that these genes on their own subtly affect the behavior. The unc-29 and acr-16 genes are expressed in the male- specific muscles We hypothesized that in the unc-29(lf);acr-16(0) mutant, signaling from circuit components that maintain vulval contact is impaired, resulting in males that cannot sustain their position at the vulva. To address which neurons and muscles were used in the male mating circuit in this step of behavior, we first determined where unc-29 and acr-16 were expressed in the male tail. In adult males, a YFP expression construct that contains a 5.3 kb sequence upstream of the acr-16 start codon (Pacr-16:YFP) was observed to PLoS Genetics | www.plosgenetics.org 3 March 2011 | Volume 7 | Issue 3 | e1001326