A practical approach to the collection, fixation, softening, and sectioning of whole honey bees (Apis mellifera) for histologic evaluation

Authors: COOK, SARAH - University Of California, Davis; Nino, Bernardo; Drouville, Laura; ALEX, CHARLES - University Of California, Davis; Seshadri, Arathi; NINO, ELINA - University Of California, Davis

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/22/2023
Publication Date: 8/14/2023
Citation: Cook, S.E., Nino, B.D., Drouville, L., Alex, C.E., Seshadri, A.H., Nino, E.L. 2023. A practical approach to the collection, fixation, softening, and sectioning of whole honey bees (Apis mellifera) for histologic evaluation. Journal of Veterinary Diagnostic Investigation. https://doi.org/10.1177/10406387231191732.
DOI: https://doi.org/10.1177/10406387231191732

Interpretive Summary: Honey bees face challenges relating to pest and disease incidences that greatly impact colony health and performance. Honey bees are generally not covered in veterinary pathology training programs. Given their unique anatomical and biological differences from the other species more traditionally evaluated by veterinary pathologists, establishing routine and consistent methods for processing samples for histology assures accurate diagnostic and research conclusions. Being the first of its kind, this study developed and tested several field protocols for collection and processing of bees. Results indicate that routine formalin fixation is adequate, and that immobilizing bees with indirect dry ice exposure aids in sample collection without negatively impacting the quality of histologic sections.

Technical Abstract: The western honey bee (Apis mellifera) is economically important as the primary managed pollinator of many agricultural crops and for production of honey, wax, and other value-added products. Honey bees are not classically or thoroughly covered in veterinary pathology training programs. Given their unique anatomical and biological differences from the other species more traditionally evaluated by veterinary pathologists, establishing routine and consistent methods for processing samples for histology assures accurate diagnostic and research conclusions. We developed and tested several field protocols for collection of bees. We compared the tissue quality outcomes for worker bees fixed, collected, and/or softened under the following protocols: 1) routine formalin fixation, 2) softening chitin via exposure to Nair for two days or 3) five days , 4) shortened times between formalin submersion and trimming of body segments to enhance penetration of formalin into internal tissues, 5) ethanol submersion of specimen prior to formalin fixation, 6) indirect dry ice exposure, and 7) prolonged -80'C storage. Routine formalin fixation, exposure to Nair for two days, indirect dry ice exposure, and trimming body segments within two hours of formalin submersion resulted in highest quality histologic tissue sections. The poorest quality sections resulted from softening of chitin for five days via exposure to Nair, submersion in ethanol for three days prior to formalin fixation, and prolonged storage at -80'C. Our results indicate that routine formalin fixation is adequate, and that immobilizing bees with indirect dry ice exposure aids in sample collection without negatively impacting the quality of histologic sections.