Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Brain Behav Immun. Author manuscript; available in PMC 2016 July 1.
Published in final edited form as:
PMCID: PMC4467992

FMRFamide signaling promotes stress-induced sleep in Drosophila


Enhanced sleep in response to cellular stress is a conserved adaptive behavior across multiple species, but the mechanism of this process is poorly understood. Drosophila melanogaster increases sleep following exposure to septic or aseptic injury, and Caenorhabditis elegans displays sleep-like quiescence following exposure to high temperatures that stress cells. We show here that, similar to C. elegans, Drosophila responds to heat stress with an increase in sleep. In contrast to Drosophila infection-induced sleep, heat-induced sleep is not sensitive to the time-of-day of the heat pulse. Moreover, the sleep response to heat stress does not require Relish, the NFκB transcription factor that is necessary for infection-induced sleep, indicating that sleep is induced by multiple mechanisms from different stress modalities. We identify a sleep-regulating role for a signaling pathway involving FMRFamide neuropeptides and their receptor FR. Animals mutant for either FMRFamide or for the FMRFamide receptor (FR) have a reduced recovery sleep in response to heat stress. FR mutants, in addition, show reduced sleep responses following infection with Serratia marcescens, and succumb to infection at a faster rate than wild-type controls. Together, these findings support the hypothesis that FMRFamide and its receptor promote an adaptive increase in sleep following stress. Because an FMRFamide-like neuropeptide plays a similar role in C. elegans, we propose that FRMFamide neuropeptide signaling is an ancient regulator of recovery sleep which occurs in response to cellular stress.

1. Introduction

An increase in sleep following an immune challenge is common to many species (reviewed in (Imeri and Opp, 2009)), including humans (Mullington et al., 2000) and Drosophila (Kuo et al., 2010). Similar to mammals, the Drosophila acute sleep response to infection is dependent on the time of day of inoculation, and requires an NFκB transcription factor, Relish (Kuo et al., 2010). A recent study shows that bacterial toxins, as well as other stressors, such as osmotic shock or heat shock, induce sleep-like quiescence in adult Caenorhabditis elegans nematodes (Hill et al., 2014). Together, these findings indicate that sleep is a conserved response to stressful stimuli. Importantly, the sleep response promotes survival (Hill et al., 2014; Kuo and Williams, 2014a), indicating that it is adaptive and beneficial to the animal.

Many components of the mammalian innate immune response, particularly pro-inflammatory cytokines, exert sleep-promoting effects likely through actions in hypothalamic nuclei (Obal and Krueger, 2003). In flies, expression of Relish in the fat body, which is a major site of immune response signaling, is necessary for the sleep promoting effects of aseptic injury and immune challenge (Kuo et al., 2010) and has a role in daily night-time sleep regulation (Williams et al., 2007). We have recently demonstrated that altering neuronal excitability in the mushroom body to manipulate sleep (Joiner et al., 2006) influences survival outcome during bacterial infections (Kuo and Williams, 2014b). Thus, communication from peripheral tissue to the brain is likely a key mechanism that underlies the injury/infection-induced sleep response in Drosophila. However, the signaling molecules and neuronal circuitry that underlie these processes are unknown.

In C. elegans, the neurosecretory ALA neuron promotes the sleep-like quiescence in response to cellular stress (Hill et al., 2014). In response to heat stress, the ALA neuron depolarizes and releases neuropeptides encoded for by the gene flp-13 (Nelson et al., 2014). FLP-13 peptides are similar to Drosophila Phe-Met-Arg-Phe-amide (FMRFa) peptides. FMRFa is a member of the FMRFa related peptide family (FaRP), which in addition to FMRFa, include dromyosuppressin, drosulfakinin, neuropeptide F (NPF), and short neuropeptide F (sNPF) (Nassel and Winther, 2010). Both NPF and sNPF have sleep-regulating roles (Chen et al., 2013; He et al., 2013; Shang et al., 2013). Each of these peptide genes encodes for one or more peptides with Fmrfa encoding for eight (Schneider and Taghert, 1990). Fmrfa is expressed in the central nervous system (CNS) as well as in thoracic neurosecretory cells (Nassel and Winther, 2010). The function of fmrfa is not fully understood, but has been reported to be important for escape responses in larval (Klose et al., 2010) and adult flies (Kiss et al., 2013). The FMRFa receptor (FR; CG2114) has varying affinity for the different FMRFa peptides and is also capable of binding Dromysosuppressin (Cazzamali and Grimmelikhuijzen, 2002; Johnson et al., 2003; Meeusen et al., 2002). FR is related to the neurotensin/thyrotropin-releasing factor receptor family in mammals (Johnson et al., 2003). Based on the fact that FLP-13 peptides promote sleep-like quiescence in response to stress in C. elegans, we tested the hypothesis that FMRFa and its corresponding receptor promote the stress-induced sleep response in Drosophila.

2. Material and Methods

2.1 Fly stocks

Flies were grown on standard dextrose-cornmeal media. Control strains included Canton-Special (CS), w1118, and yellow, white (y,w). Other strains were obtained from the Bloomington Drosophila Stock Center (Indiana University), and include Mi{ET1}FMRFaRMB04659, FMRFaRJF01879, Df(3L)BSC428, and P{SUPor-P}FMRFaKG01300. RelishE20 mutants (Hedengren et al., 1999) were isogenized to CS as previously described (Williams et al., 2007).

2.2 Behavioral Assays

Locomotor activity and sleep were measured as described previously (Kuo et al., 2012). Briefly, female flies that were 1–3 days of age were loaded into glass activity tubes (65 mm in length) containing 5% sucrose, 2% agar medium at one end and plugged with cotton yarn at the other end. Glass tubes were loaded into Drosophila Activity Monitors (DAM2, Trikinetics, Inc., Waltham, MA), and activity was recorded for up to 7–10 days. Monitors were placed in incubators kept at 25° C in 12h:12h light: dark cycles or constant light and 50% humidity. In this assay, activity counts correspond to breaks of an IR beam that bisects the tube. Sleep is defined as an activity count of zero for a minimum of 5 consecutive minutes (Huber et al., 2004). Data were processed using custom software, Insomniac 3 (written in MSCV6, by Thomas Coradetti).

Flies were exposed to heat shock by transferring monitors to an incubator in the same light phase, but set to a temperature of 37° C. After one hour, flies were returned to the 25° C incubator. Monitors containing handled control flies were briefly removed from the 25° C incubator, but immediately returned without exposing to a heat pulse. Sleep responses were quantified as described previously (Kuo et al., 2010). Briefly, differences between minutes of sleep in a given time increment following treatment (heat pulse or infection) and the equivalent baseline time increment were calculated. This difference was subtracted from an average difference for equivalent time points obtained from a handled control group, where available. Flies that died within 24 h after treatment were excluded from the analysis.

2.3 Bacterial Infection

Serratia marcescens (ATCC #8100) were grown overnight in LB medium to saturating concentrations. The next day, the culture was diluted in phosphate buffered saline containing 1% food coloring (Brilliant Blue FCF) to an O.D.600 of 0.03. Three to four days after flies were loaded into monitors, they were removed from the incubator, anesthetized with CO2 and the diluted bacterial solution was manually injected using glass capillary needles (WPI, Inc., #1B100F-4), as described previously (Kuo et al., 2012). The monitors were then returned to the incubator, and flies were recorded for an additional 5–10 days to monitor survival. Survival was determined using the Insomniac 3 software. A fly was considered dead when activity counts remained at zero for at least 24 hours.

Sleep measurements in flies excluded those that died within the first 24h after inoculation. Due to technical difficulty in performing manual injections, handled control groups were not included in these assays in order to minimize the amount of time that monitors were outside the recording chamber. Changes from baseline sleep were therefore calculated as described above, except values were not normalized to a handled control group.

2.4 Statistical Analyses

Student’s unpaired t-test was used to compare sleep parameters between indicated groups. A Bonferroni correction was applied to p-values where appropriate. One-way ANOVA with Tukey’s post-hoc was used to measure differences between three or more groups. A two-way ANOVA was used to evaluate effects of time of heat pulse and genotype on sleep where indicated. Survival analyses were conducted as described previously (Kuo and Williams, 2014a). Briefly, the Kaplan-Meier estimator was used to calculate survival probability and a log rank test was used to evaluate differences between survival curves. All statistical analyses were performed using public-accessed software (PAST; (Hammer et al., 2001).

3. Results

3.1 Heat stress induces sleep

We first determined the effects of heat stress (HS) on sleep in wild-type animals. Canton-S and w1118 wild-type flies were placed at 37° C for 1 h (see Methods) at zeitgeber time (ZT) 18, which is 6 h into the dark phase of a 12h: 12h light: dark cycle. This time point was chosen based on findings that infection and aseptic injury at ZT18 induce robust effects on sleep (Kuo et al, 2010). The HS flies exhibited increases in sleep relative to handled controls (HC) in the morning hours after HS, a time that coincides with lights-on and a typical period of arousal in handled control or undisturbed flies (Figure 1A). Specifically, a consistent and robust increase in sleep was observed from ZT0-6 following HS in both wild type strains (Figure 1B). Increased sleep was also observed during the second half of the day in w1118 flies, from ZT 6–12, albeit to a lesser extent than at ZT0-6 (Figure 1B).

Figure 1
Heat stress induces daytime sleep

We considered the possibility that the increased activity and concomitant sleep loss caused by the elevated temperature at night resulted in a homeostatic sleep rebound response the following morning. To address this concern, we subjected w1118 flies to a temperature of 31°C or 34° C for 1h at ZT 18. Both temperatures disturbed sleep as determined across a 4h period (ZT 16–20; Supplementary Figure 1). Despite exhibiting similar amounts of lost sleep to HS at 37° C, flies exposed to the lower temperatures did not sleep more than handled controls during the day after the heat pulse. Specifically, after HS at 31° C, flies showed a net change of sleep that was less than baseline (−29.6 ± 7.9 minutes from ZT 0–6, p< 0.002, one-way t-test, n=16), and no change when flies were exposed to 1 h at 34 ° C (−2.1 ± 4.3 minutes from ZT 0–6, p=0.62, one-way t-test, n=48). Therefore, the HS-induced sleep response is due to the stress of the increased temperature and is not (solely) due to the incurred sleep debt.

To evaluate potential time-of-day effects of HS, we compared the effects of HS at ZT18 (during the middle of the night period) to the effects of HS at ZT0 (lights on), ZT6 (during the middle of the day period), and ZT12 (lights off). All four time points elicited significantly increased sleep over the following 24 h in both w1118 and CS flies (Figure 1C). A modest but significant time-of-day effect was detected in w1118 flies (p< 0.02, one-way ANOVA; Figure 1C). This was attributed to a weaker response when flies were subjected to HS at ZT 12 as compared to that at ZT 18 (p< 0.01, Tukey’s post-hoc). No significant time-of-day effect was observed in CS flies (p=0.2, one-way ANOVA; Figure 1C). These data indicate that, unlike infection-induced sleep, HS-induced sleep is not strongly dependent on the time-of-day of the heat pulse. However, we noted that the increase in sleep measured across the 24h was largely attributed to an increase in daytime sleep (Figure 1A), leaving open the possibility that the circadian clock may determine the distribution of increased sleep across the day. The variations in responses of w1118 flies in comparison to CS indicate an influence of the genetic background, as previously described for other sleep measurements (Zimmerman et al., 2012).

Temperature is a known zeitgeber in Drosophila (Glaser and Stanewsky, 2007). Even mild and brief heat pulses are capable of producing a phase shift in locomotor activity rhythms in flies in a manner similar to that of a light pulse (Sidote et al., 1998). Although HS was applied at times of day that are not associated with producing significant shifts in activity phase (Dushay et al., 1990), we nonetheless sought to determine whether the HS-induced sleep in the morning is the result of a temporary phase shift. We therefore subjected flies to HS in the absence of a functioning circadian clock, in constant light. Constant light (LL) degrades integral clock genes in flies and renders them arrhythmic (reviewed in (Devlin and Kay, 2001). In the LL condition, HS increased sleep immediately following the heat pulse (Supplementary Figure 2). This indicates that a phase shift cannot account for the increase in subsequent morning sleep.

3.2 Heat stress induced sleep does not require NFκB

The sleep response to septic/aseptic injury requires the NFκB transcription factor Relish (Kuo et al., 2012; Kuo et al., 2010; Kuo and Williams, 2014a), which is central to the Drosophila innate immune response (Hedengren et al., 1999). We therefore tested the hypothesis that Relish is generally required for other stress-induced sleep responses including that induced by heat stress. This hypothesis predicts that genetic absence of Relish function would impair HS-induced sleep. We tested this prediction by subjecting RelishE20 null mutants to HS at ZT 18. Surprisingly, RelishE20 mutants showed a robust induction of sleep in response to HS at ZT 18 (Figure 2B). In comparison to CS wild-type controls, the increase in sleep extended beyond the daytime, to at least ZT 18 of the following night (Figure 2C). Therefore, genetic pathways distinct from Relish regulate HS-induced sleep. This indicates that sleep induced by different types of stress is regulated by distinct mechanisms.

Figure 2
Heat stress induces sleep in RelishE20 mutants

3.3 FR deficient flies have a reduced sleep response to HS

Because FMRFamide-like peptides encoded by the gene flp-13 are required for the sleep-like response to heat stress in C. elegans (Nelson et al., 2014), we tested whether the Drosophila FMRFa receptor, FR, regulates HS-induced sleep in flies. We compared the HS-induced sleep responses of flies containing a transposable element, minos (Metaxakis et al., 2005) inserted into the FR gene, FRMB04659, to w1118 flies, which is the genetic background strain for this mutant (Bellen et al., 2011). The minos insertion in FRMB04659 flies is located in the coding region, which is contained within a single exon of the FR gene and spans nucleotides 3,008,746 to 3,010,395 on the left arm of chromosome 3. This insertion predicts the removal of 447 nucleotides of coding sequence from the 3′ end of the gene, affecting all isoforms of FR. The program SMART (Schultz et al., 1998) predicts that this transposon insertion results in a truncated protein containing only four of the seven transmembrane domains and removes the intracellular G-protein coupling domain. Thus, this allele likely represents a null mutation in FR.

Homozygous FR mutants and w1118 wild-type controls were subjected to HS at ZT 18. As compared to w1118controls, HS-induced sleep was significantly reduced in FRMB04659 flies (Figure 3A and 3B). HS-induced sleep was also reduced in FR mutants when the HS was applied at ZT6 (Supplementary Figure 3A), suggesting that time-of-day effects are unlikely to explain the FR reduced HS-induced sleep phenotype. There was no difference in baseline daytime or nighttime sleep between genotypes (p=0.75 and 0.46, respectively; t-test; n=54 w1118 and 61 FRMB04659 flies). While daytime waking activity rate was also unchanged (p=0.25), FRMB04659 flies showed a modest but significant decrease in average nighttime waking activity (2.07 ± 0.05 activity counts/waking minute in FRMB04659 flies versus 2.3 ± 0.07 activity counts/waking minute in w1118 flies; p < 0.01, t-test). Interestingly, waking activity rates increased during the HS in both groups. In w1118 flies, waking activity rate remained elevated through the next day, but in FRMB04659 flies, it did not (Supplementary Figure 3C). Together, these data indicate that while FRMB04659 flies show normal sleep behavior at baseline, HS-induced behavior is disrupted at multiple levels.

Figure 3
Heat stress-induced sleep is decreased in FR mutants

In the course of performing these behavioral experiments, we noted that HS was partially lethal in FRMB04659 flies, such that about 25% of flies were killed by HS, whereas survival was nearly 100% in w1118 flies (Supplementary Figure 3B). Therefore, FRMB04659 flies are defective both for HS-induced behaviors and for survival after heat stress exposure.

To determine whether the effect of the Minos-element insertion was attributed to a lesion in the FR chromosomal locus, FRMB04659 flies were crossed with those carrying a small chromosomal deficiency that included the FR locus, Df(3L)BSC428. FRMB04659 and Df(3L)BSC428 flies were also crossed to the w1118 background to obtain heterozygous offspring. Both FRMB04659/+ and Df(3L)BSC428/+ heterozygous mutants showed significantly reduced sleep responses to HS at ZT 18 as compared to w1118 wild type controls (Figure 3B), indicating that the phenotype associated with both mutants is dominant. HS-induced sleep in the FRMB04659/Df(3L)BSC428 transheterozygotes was no different from the heterozygous mutants; the lack of an additive effect of the two mutants indicates that the phenotypes are likely attributed to lesions of the FR locus. HS-induced sleep was reduced in all genotypes as compared to the w1118 controls (Figure 3B).

The Minos element also contains a sequence that codes for GAL4, a transcription factor from yeast that can be used to activate a UAS promoter when expressed as a transgene in Drosophila (Brand and Perrimon, 1993). Expression of GAL4 is controlled by the gene in which Minos inserted, in this case the gene FR. We made use of the GAL4/UAS system to knock down FR using RNA interference specifically in cells expressing FR. We crossed FRMB04659 flies with those carrying a UAS-RNAi transgene directed against FR, FMRFaRJF01879 (UAS-RNAi-FR). Both parent lines were also crossed to w1118 background controls, and all offspring were subjected to HS at ZT 18. FRMB04659>UAS-RNAi-FR flies showed significantly reduced responses to HS as compared to both parent lines (Figure 3C and 3D). Together, these findings support a role of FR in stress-induced sleep.

FR is activated by FMRFa neuropeptides (Cazzamali and Grimmelikhuijzen, 2002; Johnson et al., 2003; Meeusen et al., 2002), which are related to nematode FLP-13 neuropeptides. We therefore evaluated responses of flies carrying a p-element inserted into the FMRFa gene, FMRFaKG01300. Homozygous FMRFaKG01300 mutants and y,w controls, which are the genetic background for these mutants (Bellen et al., 2004), were subjected to HS at ZT 18. y,w flies responded to HS by increasing sleep from ZT 0–4. Thus, y, w flies were less sensitive than w1118 and CS flies to heat-shock, consistent with the notion that sleep measurements in Drosophila are sensitive to genetic background (Zimmerman et al., 2012). Nonetheless, the FMRFa KG01300 mutant flies showed significantly reduced sleep responses in comparison to the y,w controls (Figure 4). These results support the hypothesis that FMRFa peptides are important for HS-induced sleep.

Figure 4
Heat stress-induced sleep is decreased in FMRFa mutants

3.4 FR deficient flies are defective in infection-induced sleep and are susceptible to bacterial infection

The experiments we performed with Relish mutants described above indicate that HS-induced sleep is controlled by a mechanism that is partially distinct from that induced by bacterial infection. We aimed to determine whether FMRFa/FR signaling is a pathway unique to HS-induced sleep or whether it is part of a mechanism common to infection- and HS-induced sleep. To test this possibility, FRMB04659 flies and w1118 control flies were subjected to infection with Serratia marcescens at ZT 18. w1118 flies showed a robust sleep response to infection at ZT 0–4, the morning after inoculation. In contrast, the sleep response to infection in FRMB04659 mutant flies was significantly reduced (Figure 5A and 5B). Survival time during infection was also severely reduced in FRMB04659 flies (Figure 5C), consistent with prior experiments showing a beneficial effect of infection-induced sleep (Kuo and Williams, 2014a). Both FRMB04659 and w1118 control flies exhibited nearly 100% survival when subjected to aseptic injury (Figure 5D), indicating that injury alone did not cause the survival outcomes during infection. These findings suggest that while the signaling events that induce sleep are different between infection and heat stress, these events converge on FR to produce a robust sleep response, and promote survival.

Figure 5
Post-infection sleep and survival is reduced in FR mutants

4. Discussion

Sleep is influenced by multiple environmental and behavioral factors, such as social experience (Ganguly-Fitzgerald et al., 2006), light stimuli (Harbison and Sehgal, 2009; Shang et al., 2011), copulation (Isaac et al., 2010), diet (Catterson et al., 2010; Keene et al., 2010; Linford et al., 2012; Thimgan et al., 2010; Yamazaki et al., 2012), and septic injury (Kuo et al., 2010; Shirasu-Hiza et al., 2007). Here we show a conserved response to heat stress, where similar to nematodes (Hill et al., 2014; Nelson et al., 2014), flies respond to heat shock with an increase in sleep predominantly in the first half of the daytime. The magnitude of heat stress-induced sleep was sensitive to genetic background, and unlike sleep induced by immune challenge (Kuo et al., 2010), it was not sensitive to the time-of-day of the heat pulse. Relish null mutants, which are defective for infection-induced sleep (Kuo et al., 2010), are not defective for HS-induced sleep; surprisingly, they show an exaggerated response to heat shock. The heat stress-induced sleep is regulated by FMRFa peptides and their receptor FR. FR is also required for the normal sleep response to an infection as well as for survival during an infection. Together, these findings indicate that while different stress modalities are transduced by distinct molecular mechanisms, these signaling events converge onto FR to promote sleep.

Temperature is a known zeitgeber in flies, such that they entrain to a diurnal temperature cycle, even in constant light (Glaser and Stanewsky, 2005; Yoshii et al., 2005). Mild heat pulses also induce phase shifts in circadian activity rhythms by suppressing expression of the clock gene, PERIOD (PER) (Sidote et al., 1998). The phase responses to heat pulses are also similar to those induced by light (Sidote et al., 1998). These findings indicate that temperature is an important environmental input to the circadian clock. However, the morning increase in sleep induced by a 37° C heat pulse is unlikely to be attributed to a phase shift in activity rhythms for the following reasons. First, heat pulse causes an enhanced sleep response even when delivered at ZT6, a time when there is a negligible circadian phase response to light (Dushay et al., 1990). Second, our experiments are performed under light:dark conditions, which results in the daily resetting of activity rhythms. Third, mild heat pulses (31° and 34° C), which is sufficient to produce phase shifts in both molecular and behavioral rhythms (Sidote et al, 1998), does not increase morning sleep. Finally, HS in the absence of the clock, under constant light conditions, elicits an enhanced sleep response immediately following the heat pulse. Together these findings indicate that the daytime increase in sleep following HS cannot be attributed to a shift in activity phase. Although the amplitude of the sleep response is not strongly altered by the time-of-day of the heat pulse, the response is primarily restricted to daytime hours. Thus a role of the clock cannot entirely be ruled out and requires further study.

FR mutants show reduced survival both in response to bacterial infection and heat stress, consistent with the notion that recovery sleep provides a beneficial function to the animal. This notion is supported by our prior observations that increasing infection-induced sleep in RelishE20 mutants improves survival (Kuo and Williams, 2014a). Whether rescuing stress-induced sleep in FR mutants produces a similar enhancement in survival requires further study. In any case, these data support the notion that sleep is an adaptive process that promotes survival.

Along with the current finding that FMRFa signaling promotes stress-induced sleep, neuropeptides are emerging as important signaling molecules with diverse roles in sleep and circadian rhythms. For example, the neuropeptide pigment dispersing factor (PDF) is involved in both arousal (Parisky et al., 2008; Sheeba et al., 2008) and clock output mechanisms (Renn et al., 1999). Recent work has shown sleep promoting roles of SIFamide (Park et al., 2014) and sNPF (Shang et al., 2013) secreted from pars intercerebralis and small ventrolateral clock cells, respectively. The precise role of sNPF, which, Like FMRFa, is a member of the FaRP family (Nassel and Winther, 2010), remains uncertain since it has also been reported to serve a wake-promoting role (Chen et al., 2013). Previous studies have shown that FMRFamide signaling is involved in regulating larval escape responses to stress, as indicated by decreased activity of FR mutants during stressful stimuli (Kiss et al., 2013; Klose et al., 2010). As previously described for larvae, our experiments reveal the adult FRMB04659 mutants have a reduced waking activity during and after heat stress. Thus the activity phenotype associated with this mutant does not correlate with the sleep phenotype. We propose that an optimal escape response to environmental stress enables animals to quickly migrate to a safer environment that would facilitate sleep. In support of this hypothesis, video observations have shown that even in the absence of environmental stress, fruit flies move to preferred areas in the recording chamber to sleep (Hendricks et al., 2000; Zimmerman et al., 2008).

While infection-induced sleep clearly requires the NFκB Relish (Kuo et al., 2010), it is likely that heat shock proteins (Hsp) mediate responses to heat stress. Hsp’s have been reported to function in sleep homeostasis, although a specific role of these molecules in sleep is not well understood. Sleep deprivation increases expression of Hsp’s in mammals (Terao et al., 2003) as well as in fruit flies (Shaw et al., 2002; Williams et al., 2007; Zimmerman et al., 2006). Flies deficient in expression of the heat shock protein Hsp83 show exaggerated responses to sleep deprivation and to have poor survival in response to a prolonged period of sleep deprivation (Shaw et al., 2002). The current study suggests that Hsp’s may function as early signaling events that promote sleep, and that the neuropeptide, FMRFa, is responsive to this signal. Alternatively, both sleep deprivation and heat shock induced sleep may allow cells to restore protein homeostasis through the action of Hsps.

Cellular stress induced quiescence occurs also in C. elegans adult animals (Hill et al, 2014). This quiescence has the sleep-like properties of cessation of feeding and locomotion, reduced responsiveness to weak stimuli, and rapid reversibility in response to strong stimuli. (Hill et al., 2014). The current study, together with work in C. elegans, supports a model of a conserved molecular mechanism for stress-induced sleep. The ALA neuron, which is receptive to epidermal growth factor (EGF) (Van Buskirk and Sternberg, 2007), is necessary for heat-stress induced sleep (Hill et al., 2014). EGF receptor signaling also promotes sleep in flies (Foltenyi et al., 2007) and mammals (Kramer et al., 2001). Whether it is also required for stress-induced sleep in insect and mammalian species will be an important topic for future study. Nelson et al (Nelson et al., 2014) further showed that the ALA neuron secretes quiescence promoting FLP-13 peptides, which share an RFamide motif with the Drosophila FMRFa peptides. In flies, an EGF receptor ligand is secreted from the brain region called the pars intercerebralis (PI) and targets receptors in the tritocerebrum to promote sleep (Foltenyi et al., 2007). Whether these cells also contain FMRFa peptides as part of a sleep promoting circuit analogous to worms is unknown. Our results indicate that FMRFa peptides acts on the G-protein coupled receptor FR to promote sleep. FR is most closely related to a group of paralogous C. elegans GPCRs encoded by the genes frpr-4, -6, -11, -12, -16, and -18 (M. Nelson and D. Raizen, unpublished observations). It remains to be determined whether or not these receptors function in nematode stress-induced quiescence. Future experiments performed in both Drosophila and C. elegans can test these specific predictions made by our model.


  • Drosophila exhibit increased sleep in response to heat stress (HS)
  • HS-induced sleep does not require the NFκB Relish
  • Mutants of FMRFamide signaling show defective behavioral responses to HS
  • FMRFamide receptor signaling promotes sleep and survival during bacterial infection

Supplementary Material


Supplementary Figure 1. Nighttime sleep is reduced by heat stress at various temperatures. Change in sleep was calculated from ZT 16–20, during which (at ZT 18) w1118 flies received 1h heat pulses at indicated temperatures. Heat pulses at all temperatures reduced equivalent amounts of sleep within the 4h time window (p > 0.1, one-way ANOVA; n= 16, 32, and 46 flies for 31°, 34°, and 37° C heat pulses, respectively).

Supplementary Figure 2. Heat stress induces sleep in constant light. A. Mean ± SEM minutes sleep per hour in constant light conditions; arrow indicates time of HS (n=32 flies both groups). B. Net change in sleep (min/6h) is plotted for indicated times post- HS (* = p < 0.00001, t-test; n=32 flies both groups).

Supplementary Figure 3. Effects of heat stress on sleep, survival, and waking activity rates. A. Net change in sleep in minutes/24h is shown for w1118 and FRMB04659 flies subject to HS at the indicated ZT times. A two-way ANOVA showed a significant effect of genotype (p<0.0001), but no significant effect of time-of-day of the heat pulse (p=0.27; n=79 and 39 w1118 and FRMB04659 flies, respectively, for HS at ZT 6; n=78 and 30 w1118 and FRMB04659 flies, respectively, for HS at ZT 18). B. Kaplan-Meier plots of survival of flies of indicated genotypes following heat stress (HS; 37° C for 1h) at ZT 18. HS had no effect on w1118 flies (p=0.51, log rank test; n=46–48 flies), but killed a small but significant number of FRMB04659 flies (p < 0.02, log rank test; n=42–48 flies). C. Net change in waking activity (average activity counts per waking minute) is plotted for 6h periods that start from the indicated ZT. HS was applied at ZT 18. *** = p < .0001; * = p < .01, Bonferroni corrected; n = 46 w1118 and 30 FRMB04659 flies.


This work was supported by NIH grants #R21NS078582 to JAW and #R01NS064030 to DMR. We thank Ewa Strus for technical support.


Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


  • Bellen HJ, Levis RW, He Y, Carlson JW, Evans-Holm M, Bae E, Kim J, Metaxakis A, Savakis C, Schulze KL, Hoskins RA, Spradling AC. The Drosophila gene disruption project: progress using transposons with distinctive site specificities. Genetics. 2011;188:731–743. [PubMed]
  • Bellen HJ, Levis RW, Liao G, He Y, Carlson JW, Tsang G, Evans-Holm M, Hiesinger PR, Schulze KL, Rubin GM, Hoskins RA, Spradling AC. The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes. Genetics. 2004;167:761–781. [PubMed]
  • Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993;118:401–415. [PubMed]
  • Catterson JH, Knowles-Barley S, James K, Heck MM, Harmar AJ, Hartley PS. Dietary modulation of Drosophila sleep-wake behaviour. PLoS ONE. 2010;5:e12062. [PMC free article] [PubMed]
  • Cazzamali G, Grimmelikhuijzen CJ. Molecular cloning and functional expression of the first insect FMRFamide receptor. Proc Natl Acad Sci U S A. 2002;99:12073–12078. [PubMed]
  • Chen W, Shi W, Li L, Zheng Z, Li T, Bai W, Zhao Z. Regulation of sleep by the short neuropeptide F (sNPF) in Drosophila melanogaster. Insect Biochem Mol Biol. 2013;43:809–819. [PubMed]
  • Devlin PF, Kay SA. Circadian photoperception. Annu Rev Physiol. 2001;63:677–694. [PubMed]
  • Dushay MS, Konopka RJ, Orr D, Greenacre ML, Kyriacou CP, Rosbash M, Hall JC. Phenotypic and genetic analysis of Clock, a new circadian rhythm mutant in Drosophila melanogaster. Genetics. 1990;125:557–578. [PubMed]
  • Foltenyi K, Greenspan RJ, Newport JW. Activation of EGFR and ERK by rhomboid signaling regulates the consolidation and maintenance of sleep in Drosophila. Nature Neuroscience. 2007;10:1160–1167. [PubMed]
  • Ganguly-Fitzgerald I, Donlea J, Shaw PJ. Waking experience affects sleep need in Drosophila. Science. 2006;313:1775–1781. [PubMed]
  • Glaser FT, Stanewsky R. Temperature synchronization of the Drosophila circadian clock. Curr Biol. 2005;15:1352–1363. [PubMed]
  • Glaser FT, Stanewsky R. Synchronization of the Drosophila circadian clock by temperature cycles. Cold Spring Harb Symp Quant Biol. 2007;72:233–242. [PubMed]
  • Hammer Ø, Harper DAT, Ryan PD. PAST: Paleontological statistics software package for education and data analysis. Palaeontologia Electronica. 2001;4:4.
  • Harbison ST, Sehgal A. Energy stores are not altered by long-term partial sleep deprivation in Drosophila melanogaster. PLoS ONE. 2009;4:e6211. [PMC free article] [PubMed]
  • He C, Yang Y, Zhang M, Price JL, Zhao Z. Regulation of sleep by neuropeptide Y-like system in Drosophila melanogaster. PLoS ONE. 2013;8:e74237. [PMC free article] [PubMed]
  • Hedengren M, Asling B, Dushay MS, Ando I, Ekengren S, Wihlborg M, Hultmark D. Relish, a central factor in the control of humoral but not cellular immunity in Drosophila. Mol Cell. 1999;4:827–837. [PubMed]
  • Hendricks JC, Finn SM, Panckeri KA, Chavkin J, Williams JA, Sehgal A, Pack AI. Rest in Drosophila is a sleep-like state. Neuron. 2000;25:129–138. [PubMed]
  • Hill AJ, Mansfield R, Lopez JM, Raizen DM, Van Buskirk C. Cellular Stress Induces a Protective Sleep-like State in C. elegans. Curr Biol. 2014;24:2399–2405. [PMC free article] [PubMed]
  • Huber R, Hill SL, Holladay C, Biesiadecki M, Tononi G, Cirelli C. Sleep homeostasis in Drosophila melanogaster. Sleep. 2004;27:628–639. [PubMed]
  • Imeri L, Opp MR. How (and why) the immune system makes us sleep. Nat Rev Neurosci. 2009;10:199–210. [PMC free article] [PubMed]
  • Isaac RE, Li C, Leedale AE, Shirras AD. Drosophila male sex peptide inhibits siesta sleep and promotes locomotor activity in the post-mated female. Proc Biol Sci. 2010;277:65–70. [PMC free article] [PubMed]
  • Johnson EC, Bohn LM, Barak LS, Birse RT, Nassel DR, Caron MG, Taghert PH. Identification of Drosophila neuropeptide receptors by G protein-coupled receptors-beta-arrestin2 interactions. J Biol Chem. 2003;278:52172–52178. [PubMed]
  • Joiner WJ, Crocker A, White BH, Sehgal A. Sleep in Drosophila is regulated by adult mushroom bodies. Nature. 2006;441:757–760. [PubMed]
  • Keene AC, Duboue ER, McDonald DM, Dus M, Suh GS, Waddell S, Blau J. Clock and cycle limit starvation-induced sleep loss in Drosophila. Curr Biol. 2010;20:1209–1215. [PMC free article] [PubMed]
  • Kiss B, Szlanka T, Zvara A, Zurovec M, Sery M, Kakas S, Ramasz B, Hegedus Z, Lukacsovich T, Puskas L, Fonagy A, Kiss I. Selective elimination/RNAi silencing of FMRF-related peptides and their receptors decreases the locomotor activity in Drosophila melanogaster. Gen Comp Endocrinol. 2013;191:137–145. [PubMed]
  • Klose MK, Dason JS, Atwood HL, Boulianne GL, Mercier AJ. Peptide-induced modulation of synaptic transmission and escape response in Drosophila requires two G-protein- coupled receptors. J Neurosci. 2010;30:14724–14734. [PubMed]
  • Kramer A, Yang FC, Snodgrass P, Li X, Scammell TE, Davis FC, Weitz CJ. Regulation of Daily Locomotor Activity and Sleep by Hypothalamic EGF Receptor Signaling. Science. 2001;294:2511–2515. [PubMed]
  • Kuo TH, Handa A, Williams JA. Quantitative measurement of the immune response and sleep in Drosophila. J Vis Exp. 2012:e4355. [PubMed]
  • Kuo TH, Pike DH, Beizaeipour Z, Williams JA. Sleep triggered by an immune response in Drosophila is regulated by the circadian clock and requires the NFkappaB Relish. BMC Neurosci. 2010;11:17. [PMC free article] [PubMed]
  • Kuo TH, Williams JA. Acute sleep deprivation enhances post-infection sleep and promotes survival during bacterial infection in Drosophila. Sleep. 2014a;37:859–869. [PubMed]
  • Kuo TH, Williams JA. Increased Sleep Promotes Survival during a Bacterial Infection in Drosophila. Sleep. 2014b;37:1077–1086. [PubMed]
  • Linford NJ, Chan TP, Pletcher SD. Re-patterning sleep architecture in Drosophila through gustatory perception and nutritional quality. PLoS Genet. 2012;8:e1002668. [PMC free article] [PubMed]
  • Meeusen T, Mertens I, Clynen E, Baggerman G, Nichols R, Nachman RJ, Huybrechts R, De Loof A, Schoofs L. Identification in Drosophila melanogaster of the invertebrate G protein-coupled FMRFamide receptor. Proc Natl Acad Sci U S A. 2002;99:15363–15368. [PubMed]
  • Metaxakis A, Oehler S, Klinakis A, Savakis C. Minos as a genetic and genomic tool in Drosophila melanogaster. Genetics. 2005;171:571–581. [PubMed]
  • Mullington J, Korth C, Hermann DM, Orth A, Galanos C, Holsboer F, Pollmacher T. Dose-dependent effects of endotoxin on human sleep. Am J Physiol Regul Integr Comp Physiol. 2000;278:R947–955. [PubMed]
  • Nassel DR, Winther AM. Drosophila neuropeptides in regulation of physiology and behavior. Prog Neurobiol. 2010;92:42–104. [PubMed]
  • Nelson MD, Lee KH, Churgin MA, Hill AJ, Van Buskirk C, Fang-Yen C, Raizen DM. FMRFamide-like FLP-13 Neuropeptides Promote Quiescence following Heat Stress in Caenorhabditis elegans. Curr Biol. 2014;24:2406–2410. [PMC free article] [PubMed]
  • Obal F, Jr, Krueger JM. Biochemical regulation of non-rapid-eye-movement sleep. Front Biosci. 2003;8:d520–550. [PubMed]
  • Parisky KM, Agosto J, Pulver SR, Shang Y, Kuklin E, Hodge JJ, Kang K, Liu X, Garrity PA, Rosbash M, Griffith LC. PDF cells are a GABA-responsive wake-promoting component of the Drosophila sleep circuit. Neuron. 2008;60:672–682. [PMC free article] [PubMed]
  • Park S, Sonn JY, Oh Y, Lim C, Choe J. SIFamide and SIFamide receptor defines a novel neuropeptide signaling to promote sleep in Drosophila. Mol Cells. 2014;37:295–301. [PMC free article] [PubMed]
  • Renn SC, Park JH, Rosbash M, Hall JC, Taghert PH. A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila. Cell. 1999;99:791–802. [PubMed]
  • Schneider LE, Taghert PH. Organization and expression of the Drosophila Phe-Met-Arg-Phe-NH2 neuropeptide gene. J Biol Chem. 1990;265:6890–6895. [PubMed]
  • Schultz J, Milpetz F, Bork P, Ponting CP. SMART, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci U S A. 1998;95:5857–5864. [PubMed]
  • Shang Y, Donelson NC, Vecsey CG, Guo F, Rosbash M, Griffith LC. Short neuropeptide F is a sleep-promoting inhibitory modulator. Neuron. 2013;80:171–183. [PMC free article] [PubMed]
  • Shang Y, Haynes P, Pirez N, Harrington KI, Guo F, Pollack J, Hong P, Griffith LC, Rosbash M. Imaging analysis of clock neurons reveals light buffers the wake-promoting effect of dopamine. Nat Neurosci. 2011;14:889–895. [PMC free article] [PubMed]
  • Shaw PJ, Tononi G, Greenspan RJ, Robinson DF. Stress response genes protect against lethal effects of sleep deprivation in Drosophila. Nature. 2002;417:287–291. [PubMed]
  • Sheeba V, Fogle KJ, Kaneko M, Rashid S, Chou YT, Sharma VK, Holmes TC. Large ventral lateral neurons modulate arousal and sleep in Drosophila. Curr Biol. 2008;18:1537–1545. [PMC free article] [PubMed]
  • Shirasu-Hiza MM, Dionne MS, Pham LN, Ayres JS, Schneider DS. Interactions between circadian rhythm and immunity in Drosophila melanogaster. Curr Biol. 2007;17:R353–355. [PubMed]
  • Sidote D, Majercak J, Parikh V, Edery I. Differential effects of light and heat on the Drosophila circadian clock proteins PER and TIM. Mol Cell Biol. 1998;18:2004–2013. [PMC free article] [PubMed]
  • Terao A, Steininger TL, Hyder K, Apte-Deshpande A, Ding J, Rishipathak D, Davis RW, Heller HC, Kilduff TS. Differential increase in the expression of heat shock protein family members during sleep deprivation and during sleep. Neuroscience. 2003;116:187–200. [PubMed]
  • Thimgan MS, Suzuki Y, Seugnet L, Gottschalk L, Shaw PJ. The perilipin homologue, lipid storage droplet 2, regulates sleep homeostasis and prevents learning impairments following sleep loss. PLoS Biol. 2010:8. [PMC free article] [PubMed]
  • Van Buskirk C, Sternberg PW. Epidermal growth factor signaling induces behavioral quiescence in Caenorhabditis elegans. Nat Neurosci. 2007;10:1300–1307. [PubMed]
  • Williams JA, Sathyanarayanan S, Hendricks JC, Sehgal A. Interaction between sleep and the immune response in Drosophila: a role for the NFkappaB relish. Sleep. 2007;30:389–400. [PMC free article] [PubMed]
  • Yamazaki M, Tomita J, Takahama K, Ueno T, Mitsuyoshi M, Sakamoto E, Kume S, Kume K. High calorie diet augments age-associated sleep impairment in Drosophila. Biochem Biophys Res Commun. 2012;417:812–816. [PubMed]
  • Yoshii T, Heshiki Y, Ibuki-Ishibashi T, Matsumoto A, Tanimura T, Tomioka K. Temperature cycles drive Drosophila circadian oscillation in constant light that otherwise induces behavioural arrhythmicity. Eur J Neurosci. 2005;22:1176–1184. [PubMed]
  • Zimmerman JE, Chan MT, Jackson N, Maislin G, Pack AI. Genetic background has a major impact on differences in sleep resulting from environmental influences in Drosophila. Sleep. 2012;35:545–557. [PubMed]
  • Zimmerman JE, Raizen DM, Maycock MH, Maislin G, Pack AI. A video method to study Drosophila sleep. Sleep. 2008;31:1587–1598. [PubMed]
  • Zimmerman JE, Rizzo W, Shockley KR, Raizen DM, Naidoo N, Mackiewicz M, Churchill GA, Pack AI. Multiple mechanisms limit the duration of wakefulness in Drosophila brain. Physiol Genomics. 2006;27:337–350. [PubMed]