Fig 4. Expression analysis of ecdysteroidogenic genes and feeding rescue experiment in ouib mutant larvae.

(A) The expression levels of ecdysteroidogenic genes in control and ouib mutant first instar larvae at 38 hours AEL measured by qRT-PCR (N = 3). (B) Immunostaining of the PG cells from control and ouib mutant first instar larvae at 36 hours AEL with antibodies against Spok (green) and Sro (magenta). Scale bar: 25 μm. (C) The transcriptional expression profiles of ouib and spok in w ¹¹¹⁸ during larval development measured by qRT-PCR (N = 3). The solid line and dashed lines indicate the expression levels of ouib and spok, respectively. (D) Feeding rescue experiments for ouib mutant (ouib ²⁹ /ouib ⁷⁴) larvae. Mutant animals fed 5β-ketodiol (5βkd) and 20-hydroxyecdysone (20E) became third instar larvae, while animals fed cholesterol (C), 7-dehydrocholesterol (7dC) and none remained first instar larvae. The lethal stages in each experimental condition were scored and shown in Table 2. Scale bar: 1 mm. Error bars indicate s.e.m. *; P<0.05, **; P<0.01 with Student’s t-test.