Abstract
The honeybee (Apis mellifera) gut microbiota serves as a model for host-microbe studies, as germ-free bees enable a relatively sterile system. Fresh gut homogenates are optimal for colonization, but immediate use may be limited by practical considerations, such as pathogen screening and the need to minimize repeated preparation for animal welfare. In this study, we systematically evaluated how short-term preservation conditions influence the colonization efficiency and structural integrity of the honeybee gut microbiota. Gut homogenates were prepared and either used immediately, refrigerated for one day or three days, or frozen for seven days with or without glycerol, before being fed to germ-free bees. Colonization outcomes were assessed by combining absolute quantification of bacterial 16S ribosomal RNA gene copy numbers with diversity indices and community composition analyses. Survival rates of inoculated bees did not differ significantly among treatments, but bees receiving inocula refrigerated for three days showed reduced gut mass, suggesting compromised microbial activity. Absolute quantification revealed that total bacterial loads were broadly maintained, yet taxon-specific responses varied, with Gilliamella showing marked sensitivity to freezing. Discrepancies between relative and absolute measurements highlighted the importance of absolute quantification for accurately evaluating colonization. Overall, inocula retained the ability to establish the characteristic core microbiota under all preservation conditions. Notably, refrigeration for one day preserved community features without any detectable differences from freshly prepared inocula, whereas longer refrigeration and freezing introduced measurable perturbations. These results provide methodological guidance for honeybee microbiota transplantation experiments and underscore the necessity of absolute quantification in microbiome research. IMPORTANCE: 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.