The non-neuronal cholinergic system, widely distributed in nature, is an ancient system that has not been well studied in insects. This study aims to investigate the key components of the cholinergic system and to identify the non-neuronal acetylcholine (ACh)-producing cells and the acting sites of ACh in the Malpighian tubules (MTs) of Mythimna separata. We found that non-neuronal ACh in MTs is synthesized by carnitine acetyltransferase (CarAT), rather than choline acetyltransferase (ChAT), as confirmed by using enzyme inhibitors and high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS). Fluorescence in situ hybridization revealed the presence of CarAT mRNA within MTs, specifically localized in the principal cells. Immunohistochemistry showed strong staining for A-mAChR, a muscarinic acetylcholine receptor, in the principal cells. Pharmacological analysis further demonstrated that ACh acts through A-mAChR in the principal cells to increase the intracellular Ca²⁺ concentration. These findings provide compelling evidence for the existence of a non-neuronal cholinergic system in the MTs of M. separata, and the principal cells play a crucial role in ACh synthesis via CarAT.

非神经元胆碱能系统在自然界中广泛分布，是一个古老的系统，在昆虫中尚未得到充分研究。本研究旨在研究胆碱能系统的关键组成部分，并鉴定非神经元乙酰胆碱（ACh）产生细胞以及ACh在粘虫马氏小管（MT）中的作用位点。我们发现 MT 中的非神经元 ACh 是由肉碱乙酰转移酶 (CarAT) 合成的，而不是胆碱乙酰转移酶 (ChAT)，这一点通过使用酶抑制剂和高效液相色谱-质谱联用 (HPLC-MS/MS) 得到证实。荧光原位杂交揭示了 MT 内存在 CarAT mRNA，特别位于主细胞中。免疫组织化学显示主细胞中 A-mAChR（一种毒蕈碱乙酰胆碱受体）的强染色。药理分析进一步证明ACh在主细胞中通过A-mAChR发挥作用，增加细胞内Ca ²⁺ 浓度。这些发现为M. separata MT中存在非神经元胆碱能系统提供了令人信服的证据，并且主细胞在通过CarAT合成ACh中发挥着至关重要的作用。