AimsTo establish the molecular tools for honeybee paratransgenesis. Methods and ResultsCommensal bacteria were isolated from two honeybees. Based on 16S ribosomal RNA sequence analysis, some isolates were identified as Fructobacillus fructosus, Lactobacillus kunkeei, Gilliamella apicola, Acinetobacter spp, Arthrobacter spp and Pseudomonas spp. Rolling circle and theta replicons were successfully introduced into F.fructosus and Lact.kunkeei. Green fluorescent protein was expressed into both species. The 73Kb Lactococcus lactis subsp. cremoris MG1363 operon encoding a cluster of five genes involved in the metabolism of galactose via the Leloir pathway was functionally expressed into a non-galactose-fermenting strain of F.fructosus enabling it to grow on galactose as a sole carbon source. ConclusionsFructophilic lactic acid bacteria, F.fructosus and Lact.kunkeei, are amenable to extensive genetic manipulations. Significance and Impact of the StudyThis is the first study demonstrating the feasibility of genetically engineering honeybee commensals, thus establishing the tools necessary for honeybee paratransgenesis.