Sensory and Insulin-Like Signals Suppress Seizures representing the effects of food, inedible food and no food on Nile Red fluorescent intensity for wild-type males (E) Representative Nile Red-stained wild-type males under the three different feeding conditions (scale bar and 23.5 mm). (F) Bar graph representing the effects of different feeding conditions on the mean number of Nile Red-stained fat droplets for wild-type males. (G) Representative Nile Red-stained Posm-12:unc-103(gf) males under the three different feeding conditions (scale bar and 23.5 mm). (H) Bar graph representing the effects of different feeding conditions on the mean number of Nile Red-stained fat droplets in Posm-12:inc-103(gf) males. For each genotype and condition, 10 males were analyzed for Nile Red staining and the p-values were determined using the Student’s t test. doi:10.1371/journal.pgen.1000117.g002 be breaking up and ingesting some bacteria, albeit at a reduced amount. However, males placed with no E. coli or aztreonam- treated E. coli shared similar phenotypes, including a pale appearance and empty intestines. To rule out that aztreonam- treated E coli are edible, we used Nile Red to visualize if internal fat stores are utilized in the three different conditions [18]. Males were grown on plates with Nile Red until L4-larval stage and then moved to one of three different feeding plates: with E. coli, inedible E. coli, or no E. coli. We then used two different standards of measurement to compare fat staining between males grown in different conditions: fluorescent intensity and the number of Nile Red-stained fat droplets. Interestingly, we found that both food- deprived males and males placed on inedible bacteria had significant increases in Nile Red intensity staining compared to well-fed males (Figure 2D-E). This result suggests that the fat regulatory mechanisms activated in food-deprived males are similarly activated in males on inedible bacteria. However, in contrast to fat droplet intensity, we found that males grown on aztreonam-treated and non-treated bacteria had significantly higher numbers of Nile Red-stained fat droplets than males grown without bacteria (Figure 2F). Therefore, although males grown on aztreonam-treated bacteria showed similar overall increases in fat- droplet intensity, our data suggests that males grown on aztreonam-treated bacteria are not mobilizing fat stores as quickly as food-deprived males. To reconcile the fact that males placed on azrtreonam-treated E. coli looked similar to starved males yet had larger numbers of fat stores, we hypothesized that sensation of bacteria could partially block fat mobilization. To test this, we transgenetically expressed a mutant gain-of-function UNC-103 K+ channel in chemosensory neurons using the osm-12 promoter [19,20], and measured effects on the number of Nile Red stained foci (Figure 2G-H). The transgenic UNC-103(gf) K+ channel contains an A331T change in the sixth transmembrane spanning domain (S6) [16,21–23]. This Posm-12:unc-103(gf) construct should hyperpolarize chemosensory neurons, reducing their ability to transduce olfactory signals. To confirm the construct reduces the function of chemosensory neurons, we verified that the animals showed previously identified chemotaxis defects (Figure S1A) [24,25]. If chemosensation of bacteria blocks fat mobilization, males expressing Posm-12:unc- 103(gf) should show a decrease in fat stores on aztreonam-treated bacteria. This was indeed the case; Posm-12:unc-103(gf) males had significantly lower numbers of fat droplets on both aztreonam- treated bacteria and on no food (Figure 2H). Although the increased Nile Red intensity and differences in the number of stained foci raise interesting questions about fat homeostasis that are beyond the scope of this work, our results suggest that males placed on aztreonam-treated bacteria are similarly food-deprived as males cultured with no bacteria. In addition to visualizing fat storage in males placed on inedible bacteria, we used GFP-expressing bacteria to visualize edibility, and also tested the ability of aztreonam-treated bacteria to sustain developmental growth. As expected, we found that untreated GFP-expressing E. coli could be visualized in the isthmus of the pharynx, the grinder, and in the intestines (Figure S1B-C). Although the worms grinded up most of the bacteria before passage to the intestines, for 95% (n = 20) of males observed, we detected small amounts of intact GFP-expressing bacteria in the intestine (n = 20). In contrast, when we treated the GFP-expressing bacteria with aztreonam, we never detected any fluorescent E. coli in the intestines (n = 20) (Figure S1C). In 55% of the males placed on inedible bacteria, single, long chains of bacteria were seen trapped in the isthmus of the pharynx, suggesting that few of the aztreonam-treated bacteria are small enough to enter the pharynx, but too large to pass through the grinder and intestines. To verify that aztreonam-treated E. coli are not digested and cannot sustain growth, we placed starved L1 larva on the three different feeding plates and visualized growth rate during our standard 15hr period. C. elegans hatch in the developmentally arrested L1 stage and only initiate postembryonic development in the presence of food (bacteria) [26–28]. Thus, we predicted that worms placed on aztreonam-treated E. coli, should arrest at L1, similar to worms placed on plates with no food. We monitored the number of gonadal cell nuclei to compare the relative developmental stage of worms grown under the three different feeding conditions (Figure S1D). We found that synchronized L1s that were placed on food for 15hrs all contained 12-celled gonads. In contrast, synchronized L1s that were placed on either aztreonam-treated bacteria or no bacteria for 15hrs were arrested, containing 4-celled gonads. Similar to worms grown without bacteria, our results suggest that worms grown on aztreonam-treated bacteria are not ingesting and breaking down nutrients required for developmental growth. Therefore, we conclude that aztreonam-treated bacteria are inedible, and the sensation of the inedible bacteria can block various food-deprivation physiological responses, including mobi- lization of fat stores and reduced muscle excitability. Chemosensory Neurons in the Head Regulate Sex-Muscle Excitability C. elegans uses chemosensation to locate the source of attractive odors (presumably for food), to avoid noxious odors, and to determine whether to enter the dauer diapause stage [29–31]. In addition, males use chemosensation to locate and mate with hermaphrodites [32–34]. To accomplish these tasks, worms use chemosensory neurons that have sensory cilia exposed to the environment, which can react to various chemicals. In regard to male mating, our finding that inedible food can partially block the suppressive effects of food deprivation suggests that chemosensory neurons down-regulate sex-muscle excitability, and their activity is attenuated when food stimulus is present. To determine if lack of sensing E. coli partially suppresses sex-muscle excitability, we assayed known chemosensory mutants. We generated double- mutants with unc-103(0) and osm-5(p813) and osm-9(ky10), and assayed the effects on muscle seizures in the three different feeding conditions (Table 1). Neither chemosensory mutation significantly affected the percentage of protracted males under the inedible- food condition; however, each mutation significantly increased the percentage under the no-food condition. These results are consistent with chemosensory neurons actively suppressing sex- muscle excitability under food-deprived conditions, and chemo- sensation of bacteria repressing the neurons’ activity. PLoS Genetics | www.plosgenetics.org 5 July 2008 | Volume 4 | Issue 7 | e1000117