Sensory and Insulin-Like Signals Suppress Seizures Figure 1. Effects of food deprivation on male sex-muscle excitability. (A) Mating success for fed and 15hr food-deprived males. Males were scored as successful if they sired at least 1 progeny. p-value determined by Fisher’s exact test (B) Graph of male muscle arecoline (ARE) sensitivity. For each concentration assayed, 20–30 males were assayed. (C) Mean sex-muscle G-CaMP responses to 10mM ARE for fed (n = 4) and food-deprived (n = 4) males. The * denotes well-fed time points that are significantly different (p#0.05, Bonferroni posttest) then the food-deprived control. For fed males, the mean dsRed intensity trace is shown as a control. Error bars represent standard error of the mean. (D) Three representative frames displaying ARE-induced calcium changes in the sex muscles of a well-fed male (time between each frame is approximately 0.7 seconds). Anterior is to the right and the dorsal protractor muscles are labeled. Scale bar 6 mm. doi:10.1371/journal.pgen.1000117.g001 conditions were not clear [1]. In food-deprived males, reduced sex-muscle excitability could be the result of two possibilities: as a response to decreased ingestion and nourishment from food, and/ or a response to decreased sensation of food in the environment. To differentiate between these two hypotheses, we asked if exposing males to inedible E. coli, i.e. bacteria they can sense but not ingest, is similar to depriving them of food. We treated E. Table 1. Chemosensory mutations block food-deprivation suppression of sex-muscle excitability. Genotype unc-103(0) osm-5(p813) osm-9(ky10) unc-103(0) osm-5(p813) unc-103(0); osm-9(ky10) % Protracted % Protracted on Inedible on Food Food % Protracted on No Food 39 (102)a 0 (10) 6 (32) 33 (163) 41 (59) 20 (85) 0 (10) 5 (20) 22 (51) 28 (40) 9 (101) 0 (10) 9 (34) 20b (96) 21b (56) aNumbers in parentheses refer to the number of animals assayed. bSignificantly different than control No Food condition p,0.01, Fisher’s Exact Test. doi:10.1371/journal.pgen.1000117.t001 coli OP50 with the antibiotic azetreonam, which inhibits cell wall septation [17], resulting in long chains of bacteria (5 to .50 mm) that are too long to fit inside C. elegans’ mouth (Figure 2A). To observe effects on wild-type mating efficiency, we separated L4 larval-stage males and placed them on one of three conditions: standard growth plates with food (untreated E. coli), with inedible food (E. coli treated with azetreonam), and with no food (no bacteria present). Interestingly, we found that wild-type males placed on inedible bacteria for 15hrs prior to the mating efficiency were more likely to successfully mate than their food-deprived counterparts (Figure 2B). In addition to measuring mating efficiency, we also measured the effects of inedible bacteria on unc-103(0)-induced seizures. The unc-103(0) deletion allele caused 38% of males to display spontaneous spicule-muscle seizures on food, whereas only 8% of males placed on the no-food condition displayed the defect. Interestingly, 20% of unc-103(0) males placed on plates with inedible bacteria had spontaneous muscle seizures, which is significantly different from both food (p = 0.007) and no-food (p = 0.017) conditions (Figure 2C and Table 1). Taken together, these results suggest that both sensation of food and ingestion of food can influence male mating behavior, possibly through regulating the excitability of the sex muscles. Although our data indicates that inedible bacteria can partially block the effects of food deprivation, it is possible that males could PLoS Genetics | www.plosgenetics.org 3 July 2008 | Volume 4 | Issue 7 | e1000117