Alternative High-Quality Hemolymph Extraction from Adult Tenebrio molitor: A Tool for Biotechnological and...

The mealworm beetle, Tenebrio molitor, is a coleopteran of importance for both immunological and biotechnological research, and it has even been considered as a potential nutraceutical. In recent years, the study of T. molitor has undergone significant development, including immune response, host-parasite interactions, and physiological approaches. However, to perform this type of study, one of the main obstacles is obtaining sufficient hemolymph and viable hemocytes. Thus, we developed a protocol for adult specimens that enables the collection of up to 300 μL of the hemolymph-anticoagulant buffer mixture per specimen, containing approximately 1.5 × 10⁵ hemocytes, with viability ranging from 85% to 90%. The technique involves a double mesothoracic puncture (DMP) and the use of a modified anticoagulant buffer that prevents hemolymph clotting, enabling continuous extravasation and ensuring high yields. Additionally, the hemocytes recovered with this protocol are intact and can be used for subsequent analysis. The hemolymph obtained using this protocol and its applications will help to better understand the processes involving hemolymph and its components in T. molitor, paving the way for further applications.