Protocol for dissecting and excising the brain of the Manduca sexta (Lepidoptera: Sphingidae) moth.

The Carolina Sphinx moth, Manduca sexta (L.) is widely used in neurobiological research, particularly those investigating the olfactory system. Due in part to similarities in brain structure and neural processing with mammals, M. sexta is regarded as a valuable model organism in neuroscience. However, despite its increasing relevance in research, detailed protocols for brain dissection in this species remain limited. This protocol provides step-by-step guidance for opening the head capsule of M. sexta and excising the brain. It visually guides the reader using annotated diagrams highlighting key anatomical landmarks, along with microscope images illustrating each major dissection step. Troubleshooting tips and suggested strategies for addressing dissection complications are included to help readers improve their technique. In addition, the protocol emphasizes precise removal of nonneural tissues, including trachea, which is particularly important for downstream applications that require high-quality, brain-specific nucleic acids and proteins. Although this protocol was specifically designed for M. sexta, it may also serve as a useful guide for brain dissections in closely related species.