Table 1_Disrupting shadow in the prothoracic gland induced larval development arrest in the fall armyworm Spodoptera frugiperda.docx

IntroductionThe juvenile hormone (JH) and 20-hydroxyecdysone (20E) are the central regulating hormones of insect development. The timing of their secretion usually leads to developmental transitions.
MethodsThe developmental transitions were evaluated via the starvation treatment and the expressions of two key metamorphosis inducing factor in Spodoptera frugiperda. Then, the main endocrine organs, including the brain–corpora cardiacum–corpora allatum and prothoracic gland, were sampled from L4-24 h and L6-24 h larvae for the RNA-seq analysis. Additionally, the critical rate-limiting enzyme of 20E synthesis, shadow, was knocked down to mimic the downregulation of 20E synthesis in the late larval instar.
ResultsThe critical weight (CW), when JH titer declines for metamorphosis, was determined be approximately L6-24 h in S. frugiperda. However, the expression of the pupal specifier Broad-Complex and the potential “metamorphosis initiation factor” Myoglianin showed a stepwise increase between L4-24 h and L6-24 h, suggesting that the developmental transitions may occur earlier. The RNA-seq analysis revealed that both 20E and JH synthesis enzymes were downregulated at the CW. In addition, strong tendencies in the expression pattern were detected among the lists of transcripts. Further knockdown of shadow induced larval development arrest and subsequent mortality, indicating that disrupting 20E synthesis before the CW is lethal. Besides, JH synthesis enzyme was down-regulated.
ConclusionThe downregulation of 20E synthesis enzymes at the CW may represent a carefully regulated event, suggesting a deceleration of larval growth and the initiation of some underlying physiological changes to set the stage for metamorphosis.

引言 保幼激素（juvenile hormone, JH）与20-羟基蜕皮酮（20-hydroxyecdysone, 20E）是调控昆虫发育的核心激素，二者的分泌时序通常决定了发育转变的发生。
方法 本研究通过饥饿处理，结合草地贪夜蛾（Spodoptera frugiperda）中两种关键变态诱导因子的表达水平，对发育转变进行评估。随后，采集四龄第24小时（L4-24 h）与六龄第24小时（L6-24 h）幼虫的主要内分泌器官——脑-心侧体-咽侧体复合体与前胸腺，进行RNA测序（RNA-seq）分析。此外，本研究敲低了20E合成的关键限速酶shadow基因，以模拟幼虫龄期后期20E合成的下调过程。
结果 本研究确定，草地贪夜蛾中触发变态发育的保幼激素滴度下降时的临界体重（critical weight, CW）约为六龄第24小时（L6-24 h）阶段。然而，蛹发育决定因子Broad-Complex与潜在"变态起始因子"Myoglianin的表达水平在四龄第24小时（L4-24 h）至六龄第24小时（L6-24 h）间呈逐步升高趋势，提示发育转变可能更早发生。RNA测序分析显示，在临界体重阶段，20E与JH合成相关酶的表达均被下调。此外，转录本列表中的基因表达模式呈现出显著的倾向性差异。进一步对shadow基因进行敲低后，幼虫出现发育停滞并最终死亡，表明在临界体重之前干扰20E合成会导致个体死亡。同时，JH合成酶的表达也出现下调。
结论 临界体重阶段20E合成相关酶的表达下调可能是一个受到精细调控的事件，这提示幼虫生长速度放缓，并启动了潜在的生理变化，为变态发育做好准备。