Three-dimensional visualization of green fluorescence protein-labelled Edwardsiellatarda in whole Medaka larvae

The invasive fish pathogen Edwardsiellatarda is common in aquatic environments and causes the environmentally and economically destructive emphysematous putrefactive disease called edwardsiellosis. In order to understand the organism's infection pathway, medaka larvae (Oryziaslatipes) were immersion-infected with E.tarda labelled with green fluorescence protein (GFP) and then visualized in three dimensions under confocal laser microscopy and light-sheet fluorescence microscopy. Confocal microscopy revealed GFP-labelled E.tarda in the mouth, head, gill bridges, gill cover, skin, membrane fin, gastrointestinal tract and air bladder, and in the caudal vein, somite veins, caudal artery and caudal capillaries. Light-sheet microscopy additionally showed GFP-labelled E.tarda in the pharyngeal cavity, muscle of the pectoral fin and cardiac atrium and ventricle. These findings suggest that during its infection of fish, E.tarda initially adheres to, and invades, the epithelial cells of the skin, gills and gastrointestinal tract (through the pharyngeal cavity); E.tarda then enters the blood vessels to access organs, including the air bladder and heart.