16S Nanopore sequencing of Lactobacillus spp. in Apis mellifera, and investigation of their bacteriocin activity

Abstract: Apis mellifera is the most common honeybee species in the world. In recent years, there have been several reports of declines in wild, and domesticated populations. Central to honeybee health are the mutualistic relationships they have with their intestinal microbiome. The Lactobacillus species living in their digestive tract assist with nutrient digestion and pathogen protection. The aim of this study was to investigate which Lactobacillus spp. were present in the intestines of subspecies A. mellifera mellifera, and A. mellifera ligustica, and if they were able to inhibit growth of the pathogen Melissococcus plutonius. The 16S rRNA gene was amplified from gDNA extracted from two complete intestines per sample, in a PCR reaction with barcoded primers. Fragments were then analyzed with nanopore sequencing. In vitro assays of catalase-treated cell-free supernatants from Lactobacillus cultures were set up against living cultures of M. plutonius on KBHI agar plates and liquid broth media, in two experiments. The same seven Lactobacillus species previously found in honeybees were confirmed to be present in the bees of this study. The ratio of species was different between individual samples, which supports earlier findings suggesting the variation is dependent on factors such as individual health, food source, and sampling season. Liquid broth in vitro assay resulted in no inhibition of early growth phase, while the last cell count measure at24 h, recorded statistically significant difference in mean values between A. apinorum and negative control (p<0.001). Further research is needed to investigate optimum conditions for inhibition.