A practical approach to the sampling, fixation, softening, and sectioning of whole honey bees for histologic evaluation

Abstract

The Western honey bee ( Apis mellifera) is economically important as the primary managed pollinator of many agricultural crops and for the production of various hive-related commodities. Honey bees are not classically or thoroughly covered in veterinary pathology training programs. Given their unique anatomic and biological differences from the other species more traditionally evaluated by veterinary pathologists, establishing routine and consistent methods for processing samples for histology ensures accurate diagnostic and research conclusions. We developed and tested several field protocols for the sampling of honey 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 2 d or 3) 5 d; 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 2 d, indirect dry ice exposure, and trimming body segments within 2 h of formalin submersion resulted in the highest quality histologic tissue sections. The poorest quality sections resulted from softening of chitin by exposure to Nair for 5 d, submersion in ethanol for 3 d before 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.

Pollen foraging preferences in honey bees and the nutrient profiles of the pollen

“…At the individual level, selective foraging depends on the ability of bees to discriminate different nectars and pollens based on multiple sensory stimuli including color, taste, tactile qualities, and odor 15 , 21 – 26 . Thus, it is apparent that pollen foragers may be able to select pollen from available resources for nutrient complementarity which can be beneficial for colonies adapting to changing resource landscapes 19 , 20 , 25 , 27 – 29 .…”

Poor Air Quality Is Linked to Stress in Honeybees and Can Be Compounded by the Presence of Disease

“…While the pathological damage from N. ceranae infections is dependable and the loads detected followed similar trends to the molecular quantification analysis, the relationships between high temperature and poor air quality with expression levels of stress genes were weak. These abiotic stressors did not result in immediate signs of pathological damage to hypopharyngeal or mandibular glands that are indicators of honeybee health [ 42 ].…”

“…We further assayed individual bees for morphological signs (histologic imaging) of stress and for pathologic lesions of N. ceranae incidence as secondary measures of the potential adverse impacts of temperature and poor air quality. Histologic imaging, which involves the microscopic evaluation of tissues and cells, has more recently gained the attention of honeybee researchers [ 37 , 38 , 39 , 40 , 41 , 42 ].…”

Special section on honey bee health and disease

“…Most of the usual laboratory techniques (e.g., PCR, bacterial culture, histopathology) can be adapted for honey bees, although some require adjustments specific to this species. 5,6,11 The scientific and general population have expressed concerns about human, livestock, and pollinator exposure to potentially harmful pesticides, especially neonicotinoids. 3,8 For this reason, much research has aimed at detecting residues and studying their effects in different species, 2,3,8 and the use of several pesticides has been prohibited in various countries over the last decade, particularly in the European Union.…”

“…Most of the usual laboratory techniques (e.g., PCR, bacterial culture, histopathology) can be adapted for honey bees, although some require adjustments specific to this species. 5 , 6 , 11 …”