To determine which chemosensory neurons regulate spicule protraction in response to food-status, we transgenetically expressed the UNC-103(gf) K+ channel in specific groups of chemosensory neurons. We first used the general chemosensory osm-12 promoter to see if it phenocopied the effects of the chemosensory mutations. We did not use the promoters for osm-5 or osm-9, since their full expression pattern requires coding sequences that could potentially interfere with the UNC-103 K+ channel fusion. When we expressed unc-103(gf) in all chemosen- sory neurons of unc-103(0) males, we found that unc-103(0)- induced seizures were no longer suppressed by food deprivation (Table 2). These results are consistent with the ability of osm-5 and osm-9 mutations to block food-deprivation suppression of seizures. Since our transgenic gain-of-function K+ channel reduced chemosensory function, we then expressed it using more restrictive promoters (Table 2). The promoters and their expression pattern confirmed by YFP reporters were as follows: Ptax-2 (AFD, AQR, ASE, ASI, ASK, AWC, BAG), Pocr-2 (ADF, ADL, ASH, AWA, PHA, PHB), and Podr-3 (AWA, AWB, AWC, PHA, PHB) [35–37]. Reducing the excitability of ocr-2-expressing neurons did not affect food-deprivation suppression of sex-muscle seizures, suggesting that the ADF, ADL, ASH, AWA, PHA, and PHB neurons are not involved. In contrast, reducing the function of either tax-2 or odr-3- expressing neurons significantly increased the percentage of starved males that had spontaneous seizures (Table 2). Since only the AWC neurons express both tax-2 and odr-3, it is likely that these neurons suppress sex-muscle excitability under food- deprived conditions. We therefore tested if the AWCs are required for food-deprivation suppression of mating behavior via Podr-3:unc- 103(gf) and through cell-ablation (Figure S1E). However, instead of increasing mating potency under food-deprived conditions, we found that removing the AWCs significantly decreased mating efficiency in well-fed males. During the assays, we noticed that AWC-ablated (genetically or with laser) males rarely encountered the hermaphrodite during the 2hr mating interval (data not shown). This finding is similar to the observations of White and colleagues, which demonstrated that the AWCs are required for male chemotaxis to hermaphrodite pheromones [32]. Therefore, it is likely that AWC functions in multiple aspects of male behavior, and its role in chemotaxis to hermaphrodites could explain why AWC-ablated males have reduced mating efficiency. Additionally, there may be other redundant neurons that can suppress mating efficiency in response to food deprivation. To test the role of the AWCs on sex-muscle excitability more directly, we ablated the neurons in unc-103(0) males and assayed the number of fed and food-deprived males with seized muscles. Similar to the Podr-3:unc- 103(gf) and Ptax-2:unc-103(gf) experiments, AWC-ablated unc- Sensory and Insulin-Like Signals Suppress Seizures 103(0) males had a significantly higher probability of having sex- muscle seizures under food-deprived conditions than the mock- ablated controls (Figure 3A). Although we cannot conclude that the AWCs are sufficient or rule out potential roles for other neurons, our data indicates that the AWC neurons regulate the male genitalia in response to food availability. Since the AWCs are required to suppress sex-muscle excitability under starved conditions, these neurons may be more active when no food is being sensed in the environment, and attenuated while food is present. Consistent with this observation, Chalasani et al [38] used the fluorescent calcium indicator G-CaMP [15] to demonstrate that the AWC neurons are activated by the removal of odorants such as isoamyl alcohol and benzaldehyde, and remain active in the absence of these chemicals. To verify that AWC responses to odourant presence and removal corresponded to the presence or absence of food in our assays, we used G-CaMP to measure the calcium activity of the AWC neurons during fed, food-deprived, and re-fed conditions (Figure 3B-D). We transge- netically expressed G-CaMP from the odr-3 promoter and used 10 well-fed adult males to measure G-CaMP fluorescence intensity after 5 hours of food deprivation, 15 hours of food deprivation, and after 5hr re-feeding. Consistent with the findings of Chalasani and co-workers [38], we found that the AWC neurons significantly increase in fluorescence intensity during the 5 and 15 hour starvation period, and then decrease back to original levels when the male is re-fed (Figure 3B-D). These results suggest that the AWC olfactory neurons are initiating signals under food-deprived conditions that lead to decreased sex-muscle excitability. The mechanism for how the AWCs communicate to the sex muscles is not clear; it is possible that they signal through cell-cell communication or through regulating the secretion of neuroactive peptides. Food Deprivation Requires the Insulin-Like Receptor, DAF-2 In C. elegans, one of the most well-studied neuropeptide receptors is the insulin-like receptor, DAF-2. Historically, DAF-2 has been well characterized for its role in regulating physiological responses to food availability, including dauer formation, life-span, and fat homeostasis [39–43]. More recently, there is growing evidence that insulin-like signaling also appears to regulate behavioral responses to food, such as male mate searching behavior [33], thermotaxis to temperatures previously associated with food [44], and associating salt with food-deprived conditions [45]. Therefore, we hypothesized that one mechanism for AWC regulation of sex- muscle excitability could be through regulating the secretion of insulin-like peptides. Table 2. Chemosensory neurons suppress unc-103(0)-seizures in the absence of food stimulus. Transgene Nonea rgEx208 [Posm-12:unc-103(gf)] rgEx234 [Ptax-2:unc-103(gf)] rgEx205 [Pocr-2:unc-103(gf)] rgEx201 [Podr-3:unc-103(gf)] Cells Affected None All ciliated neurons AFD, AQR, ASE, ASI, AWC, BAG ADF, ADL, ASH,AWA, PHA, PHB AWA, AWB, AWC aAll genotypes contain unc-103(0). bNumbers in parentheses refer to the number of animals assayed. cSignificantly different than the non-transgenic no-food condition p,0.01, Fisher’s Exact Test. doi:10.1371/journal.pgen.1000117.t002 % Protracted on Food 30 (56)b 33 (55) 36 (89) 38 (47) 35 (37) % Protracted on Inedible Food % Protracted on No Food 15 (34) 21 (34) 24 (41) 23 (30) 22 (36) 3 (63) 18c (89) 21c (56) 9 (32) 23c (43) PLoS Genetics | www.plosgenetics.org 6 July 2008 | Volume 4 | Issue 7 | e1000117