Functional assessment of the vascular and translaminar systemicity of insecticides in a cucumber-cotton aphid model

It is difficult to control insect pests with piercing-sucking mouthparts such as aphids due to their concealed feeding behavior and limited exposure to foliar-applied insecticides. Although systemic insecticides with internal translocation through plant tissues provide a practical alternative to contact-based strategies, they often suffer from uneven deposition and behavioral avoidance by pests. Furthermore, conventional assays used for investigating systemicity provide useful information on insecticide performance; however, their outcomes can reflect a combination of systemic translocation and other exposure-related factors, such as contact toxicity, phytotoxicity, and behavioral responses, thereby making it difficult to attribute effects to a specific mode of delivery. Therefore, to complement these approaches with more functionally resolved methods, we developed an integrated bioassay framework comprising a vascular sap-feeding assay to determine insecticide delivery through plant vascular tissues by providing extracted plant sap as the sole feeding source. In parallel, a translaminar gel-to-leaf assay based on κ-carrageenan ensured uniform and localized delivery of insecticides to evaluate their ability to penetrate leaf tissues. By incorporating phytotoxicity imaging based on the excess green index and chemical residue analysis (LC-MS/MS, GC-MS/MS), we could distinguish functional systemicity from superficial or edge-related exposure. Using a cucumber-cotton aphid model, we demonstrated that neonicotinoid and pyridazine pyrazolecarboxamide insecticides exhibited clear vascular and/or translaminar activity, whereas organophosphate, pyrethroid, and avermectin insecticides exhibited limited systemic delivery under the tested conditions. Overall, this integrated framework enables a more reliable interpretation of systemic insecticide performance by distinguishing vascular transport and translaminar penetration from phytotoxicity- or contact-driven effects.