Investigating the Role of ventral veins lacking in the Endocrine Regulation of Metamorphic Timing

6 Introduction 7 Regulation of endocrinology in insect development 7 The roles of critical weight and threshold size on the onset of metamorphosis 9 Critical weight 9 Threshold size 10 Juvenile hormone and prothoracicotropic hormone 11 Halloween genes and their role in the regulation of ecdysone biosynthesis 12 The significance of Vvl, a POU transcription factor 13 Oenocytes as a site of vvl expression 15 Objectives and hypothesis 15 Gene silencing using RNA interference (RNAi) 16 Tribolium castaneum and Manduca sexta as model organisms 17 Materials and Methods 19 Animal husbandry 19 Isolating RNA and synthesizing complementary DNA (cDNA) 19 Polymerase chain reaction (PCR) and gel electrophoresis 20 Cloning using TOPO TA and synthesizing double-stranded RNA (dsRNA) 21 Injecting double-stranded RNA (dsRNA) into Tribolium and treatment of 20E 22 Quantitative RT-PCR 22 Manduca body dissections 24 Results 25 vvl expression in Tribolium 25 vvl-knockdown results in reduction of jhamt3 expression 27 vvl-knockdown results in reduction of kr-h1 expression 29 vvl-knockdown larvae exhibit decreased ecdysone-response gene expression 31 vvl-knockdown influences the expression of certain Halloween genes 33 Effect of starvation on vvl expression in Manduca 40