Exploring Honeybee Abdominal Anatomy through Micro-CT and Novel Multi-Staining Approaches

SIMPLE SUMMARY: Apis mellifera or western honeybees are insects belonging to the Order Hymenoptera and the most important pollinators worldwide with great implications in natural biodiversity and agriculture due to their importance in pollination and honey production. The characterization of honeybee anatomy with precise tools will allow a better comprehension of the physiology of these insects under different biological conditions. Here, we employed Micro-computed tomography and novel staining methods to define the morphoanatomical characteristics of the worker honeybee abdomen. We defined the 3D and 2Ds structures of the midgut and hindgut and discovered a new cell type called ventricular telocyte, with possible roles in honeybee epithelium maintenance. Overall, we propose that this method will be useful for further investigation of the structure of the honeybee abdomen under a wide variety of environmental conditions. ABSTRACT: Continuous improvements in morphological and histochemical analyses of Apis mellifera could improve our understanding of the anatomy and physiology of these insects at both the cellular and tissue level. In this work, two different approaches have been performed to add new data on the abdomen of worker bees: (i) Micro-computed tomography (Micro-CT), which allows the identification of small-scale structures (micrometers) with adequate/optimal resolution and avoids sample damage and, (ii) histochemical multi-staining with Periodic Acid-Schiff-Alcian blue, Lactophenol-Saphranin O and pentachrome staining to precisely characterize the histological structures of the midgut and hindgut. Micro-CT allowed high-resolution imaging of anatomical structures of the honeybee abdomen with particular emphasis on the proventriculus and pyloric valves, as well as the connection of the sting apparatus with the terminal abdominal ganglia. Furthermore, the histochemical analyses have allowed for the first-time description of ventricular telocytes in honeybees, a cell type located underneath the midgut epithelium characterized by thin and long cytoplasmic projections called telopodes. Overall, the analysis of these images could help the detailed anatomical description of the cryptic structures of honeybees and also the characterization of changes due to abiotic or biotic stress conditions.