Insect molting and metamorphosis are controlled by the molt stimulating
hormone ecdysone. A recent study suggests that reduced tissue oxygenation correlates with the size-sensing mechanism responsible for triggering molting. When reared in
hypoxia, larvae of Manduca sexta and Drosophila melanogaster initiate molting at lower weights than do larvae reared in normoxia. Furthermore, in Drosophila, the signaling gas
nitric oxide (NO) appears to be required for normal developmental timing. As observed in Drosophila, NO signaling targets the
nuclear hormone receptor beta fushi tarazu
transcription factor 1 (βFTZ-F1) through activation of Drosophila
hormone receptor 3 (DHR3), two key regulators of
ecdysone production and metamorphic tissue progression. We set out to directly examine the effects of
hypoxia and NO on
ecdysone secretion using prothoracic glands from feeding fifth (last) larval stage M. sexta. Our results indicate that in vitro treatment of prothoracic glands with
hypoxia (2%
oxygen) or the NO donor
DETA-NONOate significantly inhibit
ecdysone secretion.
Protein markers of glandular activity were also in keeping with an initial inhibition, measured a decrease in phosphorylated ERK (
extracellular signal regulated kinase) and an increase in non-phosphorylated 4EBP (
eukaryotic initiation factor 4E binding protein). Additionally, gene expression levels of Manduca
hormone receptor 3 (mhr3), βftz-f1,
nitric oxide synthase (nos), and the PTTH receptor torso, were quantified using real-time PCR. NO treatment increased mhr3 expression and decreased nos expression.
Hypoxia increased mhr3 transcription after 2 hr, but decreased transcription after 12 hr, with no effect on nos expression. Both NO and
hypoxia had small effects on βftz-f1 expression, yet strongly increased torso transcription. Our results demonstrate that, in isolated prothoracic glands,
hypoxia and NO signaling directly inhibit
ecdysteroid secretion, but at the same time alter aspects of prothoracic gland function that may enhance secretory response.