Fig. 6

PGK1 knockdown at the wandering stage delays metamorphosis. A Morphology, HE staining, and caspase-3 location in the fat body after dsGFP and dsPGK1 knockdown. PGK1 knockdown (500 ng/larva administered in the sixth-instar at 72 h, three times over 24 h intervals). Images were obtained after the first RNAi exposure for 60 h. dsGFP was used as a negative control. Rabbit polyclonal antibodies against caspase-3 were used as the primary antibody. Green fluorescence indicates caspase-3 presence. Nuclei were stained with DAPI (blue). A’ Quantification of fluorescent caspase-3 in A. **p < 0.01, n = 3. B TEM analysis of the fat body from A. LD indicates lipid droplets. Red arrows indicate autolysosomes or autophagosomes. Blue arrows represent the apoptotic nuclei. B’ Quantification of autophagosomes, autolysosomes, and apoptotic nucleus in B. *p < 0.05, n = 3. C and C’ The levels of ATG8-II and cleaved-caspase-3 in the fat body based on A. Western blot detection using anti-ATG8 and anti-caspase-3 antibodies. *p < 0.05, n = 3. D and D’ PGK1 acetylation levels in dsGFP and dsPGK1 fat body from A. PGK1 proteins from the fat body were purified by CNBr-activated Sepharose 4B with anti-PGK1 monoclonal antibody. *p < 0.05, n = 3. E Interference efficiency analysis of PGK1 at protein levels from the input of D. *p < 0.05, n = 3. F The Vmax of 3-PG production was determined from A in vitro. PGK1 proteins from the fat body were purified by CNBr-activated Sepharose 4B with the anti-PGK1 monoclonal antibody. n = 3. G and H Concentrations of glucose (G) and lactate (H) in H. armigera hemolymph from A. n = 3. I Phenotypes after PGK1 knockdown as A (500 ng/larva administered in sixth-instar at 72 h, three times over 24 h intervals). Images were obtained after the first RNAi exposure for 80 h. I’ Percentages of phenotypes in I based on three repeats (thirty larvae for each repeat). *p < 0.05, **p < 0.01, n = 3. J The pupation time from 6th-6 h to pupa 0 d after RNAi exposure in I. *p < 0.05, n = 3. K The average body weight of pupa after RNAi exposure in I. n = 3. Bars indicate means ± SD for three independent experiments with five larvae per replicate. Statistical analyses were performed using two-tailed Student’s t tests (*: p < 0.05 and **: p < 0.01)