Impact of dietary conditions on the survival of Treh mutant larvae. (A) Treh mutants are sensitive to low-glucose and low-protein dietary conditions. Percentages of homozygous mutant pupae were determined by the ratio to heterozygous mutants in each vial. (B) Food compositions used in (A and D). Values are g per 100 ml. (C) The amounts of trehalose and glucose were analyzed in early-third instar larvae. Each value was normalized by protein levels and further normalized according to the level in the control larvae. Values shown are means and SD. (D) The pupariation rate of Tps1 and Treh single mutants and Tps1, Treh double mutants under various dietary conditions. Overaccumulation of trehalose rather than the loss of Treh causes deleterious effects on the adaptation to poor food conditions. Tre, trehalose; Suc, sucrose. (E) Treh mutants exhibit normal feeding behaviour. Food intake levels are evaluated by the rate of blue food ingestion at early third-instar larvae. (F) Treh mutants retain the epithelial integrity in the midgut. Leakage of ingested blue food into body cavity was tested. (G) Treh mutants have the normal segmented intraluminal pH zones in the midgut. Mid third instar larvae were fed with food containing phenol red dye to detect luminal pH. Phenol red changes from red to yellow at pH < 3 (acid zone) and to dark pink at pH > 10 (base zone). MT, Malpighian tubules. (H) Treh mutants exhibit no abnormality of autophagy in the fat body under fed and starved conditions. Dissected fat body at early third instar larvae were stained for LysoTracker (red) and Hoechst (blue) to label nuclei. Scale bar = 50 μm. n = 6~12 (A,D), n = 4 (C), or n = 10 (E). **p < 0.01, *p < 0.05; (A,C,D) one-way ANOVA with Tukey’s post hoc test, (E) two-tailed Student’s t-test.