Abstract
Post-embryonic development and molting in insects are regulated by endocrine changes, including prothoracicotropic
hormone (PTTH)-stimulated ecdysone secretion by the prothoracic glands (PGs). In Lepidoptera, two
pathways are potentially involved in PTTH-stimulated ecdysteroidogenesis, mitogen-activated protein kinase
(MAPK) and phosphoinositide 3-kinase/protein kinase B/target of rapamycin (PI3K/Akt/TOR). We investigated
the potential roles of both these pathways in Heliothis virescens ecdysteroidogenesis. We identified putative
proteins belonging to MAPK and PI3K/Akt/TOR signaling cascades, using transcriptomic analyses of PGs from
last (fifth) instar larvae. Using western blots, we measured the phosphorylation of 4E-BP and S6K proteins, the
main targets of TOR, following the in vitro exposure of PGs to brain extract containing PTTH (hereafter referred
to as PTTH) and/or the inhibitors of MAPK (U0126), PI3K (LY294002) or TOR (rapamycin). Next, we measured
ecdysone production, under the same experimental conditions, by enzyme immunoassay (EIA). We found that in
Heliothis virescens last instar larvae, both pathways modulated PTTH-stimulated ecdysteroidogenesis. Finally, we
analyzed the post-embryonic development of third and fourth instar larvae fed on diet supplemented with rapamycin,
in order to better understand the role of the TOR pathway in larval growth. When rapamycin was
added to the diet of larvae, the onset of molting was delayed, the growth rate was reduced and abnormally small
larvae/pupae with high mortality rates resulted. In larvae fed on diet supplemented with rapamycin, the growth
of PGs was suppressed, and ecdysone production and secretion were inhibited. Overall, the in vivo and in vitro
results demonstrated that, similarly to Bombyx mori, MAPK and PI3K/Akt/TOR pathways are involved in PTTH
signaling-stimulated ecdysteroidogenesis, and indicated the important role of TOR protein in H. virescens systemic
growth.
