Evaluating preservation effects on honeybee gut microbiota inocula

The gut microbes of honeybees are essential for their nutrition, immunity, and overall health. To study these microbes, scientists often use germ-free bees colonized with gut communities from donor bees. Fresh preparations are ideal, but repeated preparation is time-consuming, requires pathogen checks, and may involve sacrificing many bees. In this study, we tested practical preservation methods and found that both refrigerated and frozen samples retained the ability to colonize germ-free bees. However, one day of refrigeration preserved microbial abundance and composition most similar to fresh samples, whereas longer refrigeration and freezing introduced detectable changes. These findings provide researchers with clear guidance for preparing standardized microbial inocula while also minimizing repeated dissection of donor bees, thereby improving both experimental reproducibility and animal welfare.