Green, Yellow, and Red Fluorescent Proteins as Markers for Bacterial Isolates from Mosquito Midguts

The growing awareness that microbial symbionts residing in mosquito midguts can interrupt transmission of vector-borne diseases has stimulated interest in understanding their potential role in mosquito biology. Fluorescent proteins are powerful molecular markers that provide for detailed analysis of the function and behavior of specific midgut bacterial isolates without disturbing the normal gut microbiota. The aim of this study was to label bacterial isolates from the midgut of Ochlerotatus triseriatus, the primary vector of La Crosse virus, with green, yellow, and red fluorescent proteins (GFP, YFP, RFP) via electroporation. We also assessed the stability of GFP-, YFP-, and RFP-bearing plasmids and their effect on bacterial growth. Seven of eleven bacterial species could not be labeled despite several attempts. Labeling of Escherichia coli and Enterobacter cloacae was successfully achieved with all three fluorescent proteins. In contrast, labeling of Aerococcus viridans was achieved with GFP only and labeling of Aeromonas hydrophila was achieved with GFP and YFP only. The stability of GFP plasmid varied among bacterial species with A. hydrophila followed by E. cloacae having the most stable GFP label. In contrast, YFP and RFP plasmids were very stable in all bacterial species possessing these labels. GFP plasmid reduced the growth of labeled strains relative to wild type but this effect was not evident in YFP and RFP plasmids. These findings suggest that some mosquito midgut bacterial isolates can effectively be labeled with GFP, YFP and RFP plasmids allowing non-destructive studies on their functions within the vector.