TC001062

Ten of our fifteen larva made it to the pupal stage. However, no pupae successfully transformed into adult beetles. Within the pupal stage we saw a reduced length to width ratio and darker colors. This is likely due to FOXO’s effects on allometry and metabolism. Additionally, there were unusual GFP signals. The transgene that causes this gene is normally only active in wing and eye tissue, but the pupae also had activity along the base of the T2 1nd T3. This could have resulted in ectopic wing or eye development on the beetle's’ back if they had successfully matured. However, no pupae developed into adult beetles. The fact that FOXO RNAi had an effect on both the wing and eye tissues supports the idea of conserved function with Drosophila, which were also affected in the eye and wing. However, with no adult beetles, we can’t be sure of the exact effects in the wing and eyes of tribolium. Additionally, since FOXO knockout wasn’t lethal in planthoppers, these findings suggest either a diverged function of the gene or off target effects from our RNAi. One potential reason why our RNAi had lethal results is FOXO’s role in the insulin pathway. The Insulin Receptor (InR) is initially activated upon binding of insulin-like peptides. The activated receptor either phosphorylates directly the enzyme phosphoinositide 3-kinase (PI3K) or acts indirectly through the insulin receptor substrate proteins Chico and Lnk. Elevated levels of PIP3 recruit the PH-domain-containing protein kinase B (PKB/AKT) to the plasma membrane, facilitating its activation by phosphoinositide-dependent kinase 1 (PDK1). PKB signals downstream by inhibiting the FOXO (forkhead box, subgroup “O”) family of transcription factors (FOXO in Drosophila). Inhibition of the IIS pathway allows FOXO to negatively regulate the expression of pro-aging genes and positively regulate the expression of anti-aging genes. In studies on drosophila, inhibition of Chico resulted in increased longevity, however, when FOXO is inhibited we saw a lethal effect. Another explanation for the lethality of our RNAi would be an unintentional off-target effect. We observed ectopic signaling in tissues on the back. The lack of functionality of foxo in these tissues is a potential explanation for our lethal results. In particular, it is shown that FOXO is able to bind to the AMP gene regulatory region to activate AMP transcription. AMP (more significantly cAMP) is critical for intracellular signal transduction. An alteration in metabolism is a potential reason for our observed phenotypes of discoloration and death. Lastly, foxo plays a role in promoting the expression of gluconeogenic enzymes that indirectly affect glucose uptake in peripheral tissues. In our mutants, there could be a lack of expression of these enzymes that are critical for cell metabolism and tissue growth. For future research, we could perform RNAi to target other key players in the insulin pathway and observe whether or not they have a similar lethal result.