Long-term fluorescence live imaging of honeybee embryos using light sheet fluorescence microscopy and halocarbon-based liquids.

The eusocial honeybee Apis mellifera is a key pollinator and model for insect development, offering insights into the evolutionary history of holometabolous insects. Honeybee embryos develop in a controlled hive environment, which has led to potential adaptations in embryogenesis compared to solitary insect species such as the fruit fly Drosophila melanogaster. However, previously applied static imaging techniques are not well-suited to study morphogenic events. Here, we combine mRNA-based transient fluorescence labeling, Perfluorodecalin as the imaging medium, and a custom sample chamber for light sheet fluorescence microscopy to enable long-term live imaging of honeybee embryos from blastoderm formation to hatching. Our approach provides the first dynamic visualization of extra-embryonic membrane formation in honeybees, which exhibits variable window closure locations at the posterior-ventral area of the embryo. This contrasts with the red flour beetle Tribolium castaneum, where the serosa window closes at a confined anterior-ventral area. Taken together, our methodological framework expands the toolkit for alternative insect models, enabling comparative studies and investigations of environmental stressors, such as pesticides, on development. Plasmids used and datasets acquired in this study are publicly available, supporting future studies on insect diversification and conservation.