In situ analysis of the bacterial community in the gut of the earthwormLumbricus terrestrisL. by whole-cell hybridization

Abstract

The bacterial community in the gut of the earthworm Lumbricus terrestris was analyzed by whole-cell hybridization with 16S rRNA targeted oligonucleotide probes. Whole-cell hybridization protocols using fluorescence-, peroxidase-, or digoxigenin-labeled oligonucleotide probes facilitated detection of significant fractions of bacterial cells stained with 4′,6-diamidino-2-phenylindole (DAPI) in the fore-, mid-, and hind-gut and cast of the earthworm. The application of peroxidase- and digoxigenin-labeled probes, however, was hampered by several methodological drawbacks: the requirement of enzymatic permeabilization, the diffuse images of stained cells, and the incompatibility with DAPI staining used as control. Quantitative analysis of the bacterial community was also influenced by its considerable variability in different individual earthworms. Though the number of bacteria detected by DAPI staining as well as by whole-cell hybridization with the fluorescent eubacterial probe Eub338 generally showed a significant increase in the number of bacteria towards the end of the gut, a decrease in bacterial numbers could be found in some earthworms. In situ analysis of the bacterial community in the fore-, mid-, and hind-gut of one individual earthworm by whole-cell hybridization with the fluorescent eubacterial probe Eub338 recorded 15, 30, and 25% of DAPI-stained bacteria, respectively. In the cast 37% of the bacteria were detected. Similar to counts obtained by DAPI and by whole-cell hybridization with probe Eub338, the number of bacteria belonging to the α-, β-, and γ-subgroups of proteobacteria increased significantly towards the end of the gut and remained high in the cast. While the most significant difference in the counts of bacteria belonging to the α-subgroup was obtained between the hind-gut and cast, bacterial populations of the β- and γ- subgroups of proteobacteria increased most prominently between the fore- and hind-gut.Key words: digoxigenin, fluorescent probes, in situ detection, Lumbricus terrestris, rRNA, whole-cell hybridization.