Quiescence Enhances Survival during Viral Infection in Caenorhabditis elegans

Abstract

Infection causes reduced activity, anorexia, and sleep, which are components of the phylogenetically conserved but poorly understood sickness behavior. We developed a Caenorhabditis elegans model to study quiescence during chronic infection, using infection with the Orsay virus. The Orsay virus infects intestinal cells yet strongly affects behavior, indicating gut-to-nervous system communication. Infection quiescence has the sleep properties of reduced responsiveness and rapid reversibility. Both the ALA and RIS neurons regulate virus-induced quiescence though ALA plays a more prominent role. Quiescence-defective animals have decreased survival when infected, indicating a benefit of quiescence during chronic infectious disease. The survival benefit of quiescence is not explained by a difference in viral load, indicating that it improves resilience rather than resistance to infection. Orsay infection is associated with a decrease in ATP levels, and this decrease is more severe in quiescence-defective animals. We propose that quiescence preserves energetic resources by reducing energy expenditures and/or by increasing extraction of energy from nutrients. This model presents an opportunity to explore the role of sleep and fatigue in chronic infectious illness.

Keywords: C. elegans; fatigue; neuropeptides; sleep; virus.

Figure 1.

Orsay virus infection induces a sleep state in C. elegans rde-1 mutants. a, b, Activity and quiescence over 8 h of individual Day 1 adult rde-1 worms uninfected (a, n = 73) or infected (b, n = 122). Each row represents an individual worm. White indicates movement and black indicates quiescence. Data are ordered from least quiescent (top) to most quiescent (bottom). Data are pooled from 12 WorMotels. c, Total time spent quiescent during the 8 h period shown in Figure 1a,b. Mean ± standard deviation. d, e, Mean bout duration (d) and number of bouts (e) related to movement quiescence bouts from data in a–c. Mean ± standard deviation. f, Feeding rate during a 10 s period: uninfected (n = 41) or infected (n = 47) rde-1 worms. Solid horizontal lines indicate medians, and dotted lines indicate first and third quartiles. g, Co-occurrence of movement and feeding quiescence, frequency table (with fraction of whole in parentheses). Movement and feeding quiescence are not independently occurring behavioral states (Χ²₍₁₎ = 63.52; p < 0.0001). ***p < 0.001, two-tailed Student's t test.

Figure 2.

Responsiveness and reversibility of Orsay-induced quiescence. a, Probability of movement following either no stimulation (Sham) or mechanical stimulation (Stim) in quiescent infected rde-1 mutants (n = 12); data are from 1 WorMotel. b1, Probability of response to mechanical stimulation in rde-1 infected worms (n = 35). Each block represents the mean probability for each response type. Data are from 1 WorMotel. b2, Probability of sustained or complex movement (category 2 or greater in Fig. 2b) following either no stimulation (Sham) or a mechanical stimulation (Stim) in rde-1 mutants. c, Latency to respond to 1-octanol olfactory stimulus in infected rde-1 mutants that were either active (n = 17) or quiescent (n = 32) at the time of the stimulus.

Figure 3.

Homeostasis and postresponse quiescence of Orsay-induced quiescence behavior. a–c, Quiescent behavior during Orsay infection in rde-1 mutants does not show homeostatic rebound. a, Average fraction of quiescent animals across 3 h period before, during, and after mechanical stimulation, with labels showing time frames used in b and c. b, The difference in the fraction of time spent quiescent in the 1 h periods during and after mechanical stimulation. c, Mean duration of movement quiescent bouts in the 1 h period before and after mechanical stimulation. Each marker shows the average bout length for an individual worm. N(un-sham) = 6, N(un-stim) = 9, N(inf-sham) = 20, N(inf-stim) = 40. Stimulation data were pooled from 2 WorMotels, and sham data were from 1 WorMotel. d, e, Orsay virus infection is associated with postresponse quiescence (PRQ) in rde-1 mutants. a, Average fraction of quiescence at 1 s time intervals relative to stimulus (or sham stimulus) for infected (N = 35) and uninfected (N = 6) animals (e.g., a value of 0.5 indicates an animal was quiescent on 50% of the trials at that specific time point). Shading indicates SEM. PRE/POST indicate time frames used in b, average change in quiescence time in seconds after stimulation (POST − PRE). Data were from 1 WorMotel. Line plots display mean ± standard error of the mean, scatterplots display mean ± standard deviation. ^nsp > 0.05; **p < 0.01; ***p < 0.001.

Figure 4.

The ALA neuron plays a larger role than the RIS neuron in promoting sleep during Orsay virus infection. a, Model of the genetic and neural control of stress-induced sleep following heat shock or ultraviolet radiation stressors. Following an acute stress exposure, EGF receptor ligand activates the ALA and RIS neurons through the EGF-receptor LET-23. RIS and ALA release somnogenic neuropeptides following activation by EGFR signaling. The carboxypeptidase E EGL-21 is required for maturation of neuropeptides. ALA and RIS neurons function partially in parallel, and ALA also activates RIS. FLP-13 neuropeptides from ALA signal through the inhibitory G-protein coupled receptor DMSR-1 to reduce activity in wake-promoting neurons. The somnogenic effects of FLP-11 require the receptors FRPR-3, NPR-4, and NPR-22. b, UV-induced quiescence requires ALA, RIS, and neuropeptide processing. Movement quiescence during 2–4 h time window following UV treatment. N = [10, 7, 9, 8], data are from 1 WorMotel. c–g, Orsay virus infection-induced movement quiescence in SIS mutants. c, Neuropeptide processing mutants [egl-21(n476); rde-1(ne219)] suppress Orsay-induced movement quiescence. N = [14, 14, 24, 25]. Data are pooled from 2 WorMotels. d, e, ALA neuron development mutants [ceh-17(np1); rde-1(ne219) (N = [31, 32, 25, 30]) and ceh-14(ch3); rde-1(ne219); N = [4, 4, 20, 20]] suppress Orsay-induced movement quiescence. Data are pooled from 3 and 1 WorMotels, respectively. f, RIS neuron development mutants [aptf-1(tm3287); rde-1(ne219)] do not suppress Orsay-induced movement quiescence. N = [24, 27, 36, 35], data are pooled from 4 WorMotels. g, FLP-11 neuropeptide mutants rde-1(ne219); flp-11(tm2706) modestly suppress Orsay-induced movement quiescence. N = [31, 28, 47, 40], data are pooled from 4 WorMotels. h, Chemogenetic inhibition of RIS suppresses movement quiescence during 2–4 h time window after UV treatment. N = [2, 3, 5, 9], data are from 1 WorMotel. i, Chemogenetic inhibition of RIS [rde-1(ne219); flp-11p:HisCl1] suppresses Orsay-induced movement quiescence. N = [8, 6, 9, 10; 63, 62, 80, 65]. Data for no histamine are from are from 1 WorMotel, and data for histamine were pooled from 8 WorMotels. Mean ± standard deviation. ^nsp > 0.05; *p < 0.05; ***p < 0.001.

Figure 5.

Feeding quiescence during Orsay-induced sleep. a, b, Feeding quiescence in SIS mutants during (a) Orsay virus-induced sleep and (b) UV stress-induced sleep. Pharyngeal pumping rate measured during 10 s interval. N(a) = [44, 50, 50, 28, 38; 50, 50, 40, 29, 44]; N(b) = [40, 25, 25, 24, 30; 30, 20, 20, 20, 30]. Mean ± standard deviation. All pairwise comparisons were made within treatment groups between experimental and control genotypes [rde-1(n219)] using Dunnett's multiple-comparisons test. ^nsp > 0.05; *p < 0.05; ***p < 0.001.

Figure 6.

Increased mortality in Orsay virus infection due to reduced quiescence. a, b, Lifespan is reduced in Orsay-infected animals compared with uninfected controls. Among Orsay-infected animals, ALA defective mutants ceh-17(np1); rde-1(n219) (a) and ceh-14(ch3); rde-1(n219) (b) has reduced lifespan compared with infected rde-1(n219). c, Among Orsay-infected animals, RIS aptf-1(tm3287); rde-1(n219) mutants do not have different lifespans compared with control rde-1(n219) animals. d, Chronic overexpression of somnogenic FLP-13 neuropeptides in Orsay virus infected ALA mutants [ceh-17(np1);rde-1(ne219); hsp-16.2p:flp-13 black-outlined triangles] restores lifespan to levels similar to control infected animals [rde-1(ne219) gray-filled circles]. Worms of all genotypes were cultivated at 25°C. e, Chronic overexpression of flp-13 causes movement quiescence. Stage 4 larvae (L4) of each genotype were incubated at 20°C (left) or 25°C (right) for 24 h and then transferred to the WorMotel as Day 1 adults and assayed for movement quiescence for 8 h at room temperature (20–22°C). Mean ± standard deviation. N = [4, 3, 19, 19], data are from 1 WorMotel. Kaplan–Meier survival curves. See Tables 4–7 for detailed statistics and sample sizes. ^nsp > 0.05; ***p < 0.001.

Figure 7.

Mechanisms of early demise in sleepless animals. a, Orsay virus load in infected animals [rde-1(n219), N = 12] and ALA mutants [ceh-17(np1); rde-1(n219), N = 12]. b, Intracellular pathogen response genes are upregulated during Orsay infection. Transcription of IPR genes increases in infected animals [rde-1(n219)] and infected ALA mutants [ceh-17(np1); rde-1(n219)] compared with uninfected animals. Two-way ANOVA with factors infection and genotype was performed separately for each gene and the main effect of Orsay infection is indicated on graph. There is neither a main effect of genotype nor an interaction effect between genotype and infection. N = 3–5 per group (see Table 3 for specifics). c, Bacteria colonize the intestine of Orsay-infected worms. GFP-expressing E. coli more rapidly infiltrate and escape the intestine of infected ALA mutants compared with infected controls. The y-axis indicates frequency for each score. For detailed statistics, see Table 8. d, Survival on UV-killed bacteria diet. Orsay-infected rde-1(n219) animals were fed either live or UV-killed GFP-expressing E. coli bacteria throughout their lifespan. Infected animals fed UV-killed bacteria had significantly increased lifespan compared with animals fed dead bacteria. Kaplan–Meier survival curve. For detailed statistics, see Table 9. e, Whole-animal ATP levels are reduced in ALA-defective animals during Orsay infection. ATP levels were not reduced in rde-1(ne219) infected animals compared with uninfected controls. ATP levels were reduced in infected ALA mutants [ceh-17(np1); rde-1(n219)] compared with uninfected animals. ^nsp > 0.05; *p < 0.05; ***p < 0.001.