Author Summary We demonstrate that lack of sensation of food in the environment can alleviate spontaneous muscle seizures via an insulin-like mediated pathway. Food restriction is known to promote many adaptive physiological respons- es, including the mobilization of fat-stores, increases in life span, and suppression of seizures. Consequently, a better understanding of the exact cellular and molecular details involved in these responses could lead to better health treatments. Here, we identify a novel mechanism activated by food restriction that occurs as a response to decreased sensation of food in the environment, rather than a response to decreased ingestion. Specifically, we show that suppression of C. elegans mutant-induced muscle seizures by food deprivation requires the activity of the AWC olfactory neurons and insulin-like signaling. We elucidate a novel role of insulin-like signaling in response to caloric restriction that does not involve the canonical FOXO/DAF-16 pathway. Instead, we propose that insulin- like control of muscle excitability activates downstream signaling molecules that include phospholipase C-c, calcium/calmodulin-dependent kinase II (CaMKII), and ether-a-go-go K+ channels. These molecules identify a novel mechanism used by organisms to regulate multiple physiological responses to changing environmental con- ditions. Results Male Mating Behaviors Are Suppressed by Food Deprivation Male mating behavior initiates when sensilla in the male tail contact a hermaphrodite. The male then presses the ventral portion of his tail against the hermaphrodite’s cuticle and begins moving backwards, keeping tail contact with the hermaphrodite and scanning for her vulva. If the male reaches the end of the hermaphrodite without sensing the vulva, he performs a ventral turn and scans the other side. Once he locates the vulva, he halts backward locomotion and attempts to insert his copulatory spicules into his mate. Spicule insertion is achieved by rapid contraction of his sex muscles, which consist of two retractor muscles and two protractor muscles attached to each of his two spicules. Once he has breached the vulva, he maintains a tonic contraction of his sex muscles, which allows sperm to be transferred into his mate [8–12]. We have found that food-deprived males are less effective at mating behavior than well-fed males. To quantify this observation, we tested the effects of food deprivation on wild-type mating success (Figure 1A). We paired both well-fed males and 15-hr food-deprived males with hermaphrodites and scored the number of males that could sire at least one progeny given a 2-hr mating interval. About 55% of well-fed males could sire at least one progeny, while only 20% of food-deprived males were successful (p = 0.001, Fisher’s exact test). The reduced mating success of food-deprived males could be due to multiple factors. Since mating behavior consists of multiple sub-steps, we focused on the spicule insertion step of mating, as inefficacy at this step is a likely reason for unsuccessful mating. Food deprivation can suppress precocious spicule protraction caused by the loss of the ERG-like K+ channel, UNC-103 (Table 1). We have previously reported that UNC-103 acts in the sex muscles to suppress muscle excitability and spontaneous spicule protraction. We reasoned that in unc-103 deletion (unc- 103(0)) males, food-deprivation could compensate for loss of the Sensory and Insulin-Like Signals Suppress Seizures gene by activating other mechanisms that reduce muscle output [1,13]. Therefore, it is possible that food deprivation reduces mating efficiency of wild-type males by suppressing pathways that increase sex-muscle excitability. Previous genetic analyses on the male circuitry have revealed that spicule muscle behaviors are regulated by at least two distinct signaling pathways [12]. Specifically, the neurons controlling sex-muscle output activate different spicule muscle ACh receptors and downstream signaling pathways to mediate distinct muscle contractile behaviors. The ACh agonist levamisole activates spicule protraction through the ryanodine receptor (RyR), UNC-68, while the ACh agonist arecoline activates spicule protraction through the L-type voltage- gated Ca2+ channel (L-VGCC), EGL-19 [12]. Other tissues and physiological responses (i.e. food availability) integrated with male mating may act on one or both of these pathways. We have recently demonstrated that food deprivation reduces unc-103(0)- mediated spicule protraction by affecting a pathway that includes CaMKII and L-VGCC/EGL-19 channels [1], suggesting that reduced mating efficiency in food-deprived males may be due to suppression of L-VGCC/EGL-19 activity. To verify in wild-type males that food deprivation results in a reduction of L-VGCC/ EGL-19-induced Ca2+ influx and muscle contraction, we tested sex-muscle response to arecoline [12]. Similar to mating success, we found that food-deprivation reduces wild-type response to arecoline-induced spicule protraction (Figure 1B). The effective concentration at which 50% of well-fed males protracted their spicules (EC50) was 21 mM while food-deprived males had an EC50 of 262 mM. In addition to measuring spicule protraction, we used the fluorescent calcium indicator, G-CaMP [15], to measure real-time calcium changes in the sex muscles of fed and food-deprived males (Figure 1C-D). G-CaMP consists of a circularly permutated enhanced GFP connected to the M13 fragment of myosin light chain kinase and calmodulin (CaM). When Ca2+ binds to the CaM of G-CaMP, CaM binds to the M13 region and the induced conformational changes result in fluorescent intensity changes [15]. We hypothesized that reduced response to ACh agonist in starved-males was due to decreased calcium signaling in the spicule muscles. To drive expression of G-CaMP in the sex muscles, we used the unc-103 1E promoter, which expresses in the spicule protractor muscles [16]. Additionally, we also co-injected dsRed as a control for intensity changes caused by reduced cell volume or unintentional background changes. As expected, we found that spicule protractor muscles of food-deprived males had significantly lower calcium responses to arecoline (Figure 1C). Figure 1C displays the averaged G-CaMP and dsRed trace for well-fed (n = 4) and the averaged G-CaMP trace for 2hr food- deprived (n = 4) males. Specifically, starved males did not show an increase in intensity in response to arecoline, while fed males showed rhythmic increases in intensity over time (Figure 1C). These observations suggest that active mechanisms in food- deprived males reduce calcium signaling and muscle contraction in response to stimulation. Taken together with previous results, we suggest that food deprivation suppresses sex-muscle excitability of both wild-type and unc-103(0) males, and the phenotype of unc- 103(0) males can be used to track how different perturbations affect this regulation. Food Deprivation Suppresses Sex-Muscle Excitability via Sensory Signals We previously identified CaMKII (UNC-43) and an EAG K+ Channel (EGL-2) as components of a signaling pathway that reduces excitability under food-deprived conditions. However, the upstream signals that activate this pathway under starved PLoS Genetics | www.plosgenetics.org 2 July 2008 | Volume 4 | Issue 7 | e1000117