The insect brain is the central part of the neurosecretory system, which controls morphology, physiology, and behavior during the insect's lifecycle. Lepidoptera are holometabolous insects, and their brains develop during the larval period and metamorphosis into the adult form. As the only fully domesticated insect, the Lepidoptera silkworm Bombyx mori experienced changes in larval brain morphology and certain behaviors during the domestication process. Hormonal regulation in insects is a key factor in multiple processes. However, how juvenile hormone (JH) signals regulate brain development in Lepidoptera species, especially in the larval stage, remains elusive. We recently identified the JH receptor Methoprene tolerant 1 ( Met1) as a putative domestication gene. How artificial selection on Met1 impacts brain and behavioral domestication is another important issue addressing Darwin's theory on domestication. Here, CRISPR/Cas9-mediated knockout of Bombyx Met1 caused developmental retardation in the brain, unlike precocious pupation of the cuticle. At the whole transcriptome level, the ecdysteroid (20-hydroxyecdysone, 20E) signaling and downstream pathways were overactivated in the mutant cuticle but not in the brain. Pathways related to cell proliferation and specialization processes, such as extracellular matrix (ECM)-receptor interaction and tyrosine metabolism pathways, were suppressed in the brain. Molecular evolutionary analysis and in vitro assay identified an amino acid replacement located in a novel motif under positive selection in B. mori, which decreased transcriptional binding activity. The B. mori MET1 protein showed a changed structure and dynamic features, as well as a weakened co-expression gene network, compared with B. mandarina. Based on comparative transcriptomic analyses, we proposed a pathway downstream of JH signaling (i.e., tyrosine metabolism pathway) that likely contributed to silkworm larval brain development and domestication and highlighted the importance of the biogenic amine system in larval evolution during silkworm domestication.
昆虫的脑是神经内分泌系统的中心，在昆虫的生命周期中控制着形态、生理和行为。鳞翅目昆虫（Lepidoptera）是全变态昆虫，其脑由幼虫态经过变态发育至成虫态。鳞翅目家蚕(Bombyx mori) 是唯一完全驯化的昆虫，在驯化过程中，经历了幼虫脑形态和一系列行为的变化。激素是昆虫生命过程中的关键调控因素。然而，保幼激素信号如何调节鳞翅目物种的大脑发育，尤其是在幼虫阶段，目前尚不清楚。我们前期工作发现保幼激素受体 Methoprene tolerant 1（ Met1）是一个候选的驯化基因，而理解人工选择如何作用于 Met1进而影响家蚕大脑和行为驯化，将为理解达尔文关于驯化的理论提供线索。该研究中，我们利用CRISPR/ cas9技术对家蚕 Met1进行敲除，发现家蚕大脑发育迟缓，该现象与表皮的提前化蛹相悖。在转录组水平上，蜕皮激素(20-hydroxyecdysone，20E)信号通路及其下游通路在突变体表皮中被过度激活，而在大脑中未出现此现象，而是与细胞增殖和特化过程相关的通路，如细胞外基质(ECM)-受体相互作用和酪氨酸代谢通路受到抑制。分子进化分析发现家蚕 Met1中一个有强烈正选择信号的氨基酸替换，该位点位于一个影响转录活性的独特基序中。体外结合活性显示家蚕Met1转录结合活性低于野蚕。与其祖先野桑蚕（ Bombyx. mandarina.）相比，家蚕MET1蛋白的结构和动态特征发生变化，在脑中的 Met1共表达网络弱化。基于整合的脑转录组比较分析，我们发现了JH下游信号通路的一个通路——酪氨酸代谢通路，可能与家蚕幼虫大脑的发育和驯化相关，同时也提出生物胺系统对于家蚕幼虫脑的驯化可能具有重要作用。.
Keywords: Artificial selection; Brain; Met1; Silkworm; Tyrosine metabolism pathway; .