Delayed pupation in Dilp2GAL4 and DdcGAL4 rescue conditions results in larger body size.

(A) From left to right: UASitpr⁺/+;Dilp2GAL4/+; P0163GAL4,itpr^sv35/ug3 (Dilp2GAL4 P0163GAL4 rescue), wild-type (Canton-S) and UASitpr⁺/+;Dilp2GAL4/+;itpr^sv35/ug3 (Dilp2GAL4 rescue) pupae. (E) Only UASitpr⁺ expression with Dilp2GAL4 in itpr^sv35/ug3 background causes a significant increase (*p<0.005; Student's t-test) in pupal length as compared to controls of all the indicated genotypes. Pupal length is restored close to wild-type in Dilp2GAL4::P0163GAL4 rescue condition. (B) From left to right: wild-type and Dilp2GAL4 rescued flies. (F) Body length of Dilp2GAL4 rescued flies is significantly more than wild-type flies (*p<0.05; Student's t-test). (C, D) From left to right: UASitpr⁺/+;DdcGAL4/+;P0163GAL4,itpr^sv35/ug3 (DdcGAL4 P0163GAL4 rescue), wild-type (Canton-S) and UASitpr⁺/+;DdcGAL4/+;itpr^sv35/ug3 (DdcGAL4 rescue) pupae (C) and flies (D). (G) Only UASitpr⁺ expression with DdcGAL4 in itpr^sv35/ug3 background causes a significant increase (*p<0.005; Student's t-test) in pupal length as compared to controls of all the indicated genotypes. Pupal length (G) and adult weight per fly (H) is increased in the DdcGAL4 rescued condition (*p<0.005; Student's t-test) but is restored close to wild-type in DdcGAL4::P0163GAL4 rescue condition. n = 10 or more for each individual genotype for (A–G). For (H), the following numbers of male flies (n) in batches of around 3 flies each were weighed for each genotype: DdcGAL4 P0163GAL4 rescue n = 26, wild-type n = 30 and DdcGAL4 rescue n = 54. (I) Total number of larvae that undergo pupation is significantly increased on introducing a prothoracic gland GAL4 (P0163GAL4) in DdcGAL4 and Dilp2GAL4 rescued conditions (*p<0.05, **p<0.005; Student's t-test). (J) Time AEL for 50% pupal formation is significantly reduced with P0163GAL4 in DdcGAL4 and Dilp2GAL4 rescued conditions (*p<0.005; Student's t-test). However the 50% pupation time in all single and double GAL4 conditions remained longer than the 50% pupation time of wild-type. For (I) and (J) 25 larvae in the following number of batches (N) were assayed for pupation for each genotype: wild-type N = 11, Dilp2GAL4 rescue N = 10, Dilp2GAL4 P0163GAL4 rescue N = 5, DdcGAL4 rescue N = 13 and DdcGAL4 P0163GAL4 rescue N = 9. (K) DdcGAL4 rescued itpr^sv35/ug3 larvae pupated earlier on being fed 20-hydroxyecdysone (∼150 hrs AEL) than larvae without 20-hydroxyecdysone (∼200 hrs AEL) (*p<0.05; Student's t-test). A minimum of 75 animals were screened in batches of 25 each. Differences in pupation rate are not apparent upon 20-hydroxyecdysone feeding in Dilp2GAL4 rescue animals due to increased lethality in this condition in late third instar larvae. The DdcGAL4 rescued condition which were not fed ecdysone, pupated at a slower rate than those observed in (J). This is very likely due to differences in culture conditions in the two cases. Results are expressed as mean±SEM.