Figure 5.

Sleep Is Required for Surviving Larval Starvation-Induced Arrest

Shown are representative experiments; additional replicates can be found in Figure S5.

(A) Adult lifespans of feeding wild-type (blue) and aptf-1(gk794) (red) worms were not significantly different (mean lifespan, N2: 14.6 days, n = 44 worms; aptf-1(gk794): 15.3 days, n = 43 worms; p = 0.71; log rank test).

(B) Lifespan of feeding wild-type (blue) and RIS(−) (yellow) adult worms shows no significant difference (mean lifespan, N2: 14.2 days, n = 94 worms; RIS(−): 14.1 days, n = 86 worms; p = 0.71; log rank test).

(C and D) No significant difference of lifespan was detected in (C) starved adult aptf-1(gk794) compared to starved N2 (mean lifespan, N2: 29.8 days, n = 60 worms; aptf-1(gk794): 29.5 days, n = 63 worms; p = 0.65; log rank test) and (D) also not in starved RIS(−) (mean lifespan N2: 32.0 days, n = 85 worms; RIS(−): 30.6 days, n = 84 worms; p = 0.06; log rank test).

(E) L1-arrested aptf-1(gk794) mutants and RIS ablated worms showed substantially reduced survival in the absence of food (time at which 50% of worms were still alive, N2: 20.7 days, n > 60 worms for each time point; aptf-1(gk794): 12.1 days, n > 60 worms; ^∗∗∗p < 0.001; RIS(−): 9.4 days, n > 60 worms; ^∗∗∗p < 0.001; significance shown from day 8; Fischer’s exact test).

(F) Arrested L1 aptf-1(gk794) and RIS-ablated worms showed a significant decline in the ability to re-enter development when fed (time at which 50% of worms were still able to recover, N2: 18.7 days, n > 60 worms; aptf-1(gk794): 11.4 days, n > 60 worms; ^∗∗∗p < 0.001; RIS(−): 8.5 days, n > 60 worms; ^∗∗∗p < 0.001; significance shown from day 8 for RIS(−) and day 10 for aptf-1 mutant; Fischer’s exact test).

(G) Sleep amount predicts starvation survival. Arrested starved L1 larvae were cultured in microchambers, and their sleep and survival were quantified. Data from individual worms within the range used for linear fitting (black line) are plotted as blue diamonds (data outside the range of the SD of the mean, including early-dying immobile individuals, were excluded; gray); R² = 0.21.

(H) Starved arrested L1 aak-1(tm1944)/aak-2(ok524)/daf-16(mgDf50)/aptf-1(gk794) quadruple mutants showed no reduced survival compared to aak-1(tm1944)/aak-2(ok524)/daf-16(mgDf50) triple mutants (time at which 50% of worms were still alive, aak-1(tm1944)/aak-2(ok524)/daf-16(mgDf50): 3.9 days, n > 60 worms; aak-1(tm1944)/aak-2(ok524)/daf-16(mgDf50)/aptf-1(gk794): 3.9 days, n > 60 worms; p > 0.05 for all days; Fischer’s exact test).

(I) FUdR-arrested L1s in the presence of food have a decreased survival. Arrested L1 survival is significantly decreased in aptf-1(gk794) and in RIS(−) worms (mean survival, N2: 25.7 days, n = 143 worms; aptf-1(gk794): 21.9 days, n = 140 worms; ^∗∗∗p < 0.001; RIS(−): 22.1 days, n = 135 worms; ^∗∗∗p < 0.001; log rank test).

See also Figures S5 and S6 and Tables S1, S2, S3, S4, and S5 for additional replicates and details on food consumption.