Department of Sciences, University of Basilicata, Via dell'Ateneo Lucano 10, 85100 Potenza, Italy.
Department of Life Sciences and Biotechnology, University of Ferrara, Via Luigi Borsari 46, Ferrara 44121, Italy.
J Insect Physiol. 2018 May-Jun;107:57-67. doi: 10.1016/j.jinsphys.2018.02.008. Epub 2018 Feb 15.
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.
昆虫的胚胎后发育和蜕皮受内分泌变化的调节，包括前胸腺激素（PTTH）刺激前胸腺（PGs）分泌蜕皮激素。在鳞翅目昆虫中，有两条途径可能参与 PTTH 刺激的蜕皮甾酮生物合成，即丝裂原活化蛋白激酶（MAPK）和磷酸肌醇 3-激酶/蛋白激酶 B/雷帕霉素靶蛋白（PI3K/Akt/TOR）。我们研究了这两条途径在棉铃虫蜕皮甾酮生物合成中的潜在作用。我们使用 PGs 的转录组分析鉴定了属于 MAPK 和 PI3K/Akt/TOR 信号级联的假定蛋白，这些 PGs 来自最后（第五）龄幼虫。使用 Western blot 技术，我们测量了 4E-BP 和 S6K 蛋白的磷酸化，这是 TOR 的主要靶标，方法是将 PGs 体外暴露于含有 PTTH（以下简称 PTTH）的脑提取物和/或 MAPK（U0126）、PI3K（LY294002）或 TOR（雷帕霉素）抑制剂。接下来，我们通过酶免疫分析（EIA）在相同的实验条件下测量蜕皮甾酮的产生。我们发现，在棉铃虫的最后一龄幼虫中，这两条途径都调节了 PTTH 刺激的蜕皮甾酮生物合成。最后，我们分析了在添加雷帕霉素的饮食中喂养的三龄和四龄幼虫的胚胎后发育，以便更好地理解 TOR 途径在幼虫生长中的作用。当雷帕霉素添加到幼虫的饮食中时，蜕皮的开始被延迟，生长速度降低，结果产生了体型异常小、死亡率高的幼虫/蛹。在添加雷帕霉素的饮食中喂养的幼虫中，PGs 的生长受到抑制，蜕皮甾酮的产生和分泌受到抑制。总的来说，体内和体外的结果表明，与家蚕一样，MAPK 和 PI3K/Akt/TOR 途径参与了 PTTH 信号刺激的蜕皮甾酮生物合成，并表明 TOR 蛋白在棉铃虫全身生长中起着重要作用。
