Carnitine acetyltransferase acts as a unidirectional compensatory enzyme for choline acetyltransferase activity in Nilaparvata lugens.

Carnitine/choline acyltransferase family facilitates acyl-group transport and regulates acetylcholine (ACh) homeostasis in insects. Five members of this family were identified in Nilaparvata lugens, among which choline acyltransferase (ChAT) and carnitine acyltransferase (CrAT) are phylogenetically closely related. Silencing ChAT or inhibiting ChAT activity by omeprazole significantly upregulated CrAT expression, whereas CrAT silencing did not affect ChAT expression. Although CrAT knockdown reduced ChAT activity in vivo, ChAT silencing did not alter CrAT activity, indicating a unidirectional compensatory relationship. Molecular docking supported choline-binding capability of CrAT, supporting its role in ACh synthesis. While CrAT silencing alone did not cause mortality, dual silencing ChAT and CrAT significantly increased insecticide susceptibility compared to ChAT silencing alone. These results demonstrate that CrAT upregulation would partially compensate for ChAT deficiency. To overcome this compensation, we screened for dual-target inhibitors and identified 5-hydroxy omeprazole, which exhibits high binding affinity for both ChAT and CrAT. Collectively, this study reveals a CrAT-mediated compensatory mechanism in insect ACh synthesis, highlighting the need to account for such adaptive responses in designing novel insecticides.