Green, yellow and red fluorescent proteins as markers for bacterial isolates from mosquito midguts

Authors: Muturi, Ephantus; Ramirez, Jose; KIM, CHANG-HYUN - University Of Illinois

Submitted to: Insects
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/31/2019
Publication Date: 2/3/2019
Citation: Muturi, E.J., Ramirez, J.L., Kim, C. 2019. Green, yellow and red fluorescent proteins as markers for bacterial isolates from mosquito midguts. Insects. 10:49. https://doi.org/10.3390/insects10020049.
DOI: https://doi.org/10.3390/insects10020049

Interpretive Summary: Bacterial communities that colonize the mosquito gut play an important role in host nutrition and reproduction and can also influence mosquito susceptibility to disease causing agents. Understanding the functions of various gut bacterial species on mosquito biology can propel discovery of bacterial species for application in mosquito-borne disease control. Conventional approach for studying bacterial functions in mosquito guts involve clearing the normal gut bacteria with antibiotic therapy administered orally and then introducing the target bacteria via blood meal or sugar meal and examining the outcome. This method has yield novel insights into how mosquitoes interact with their gut bacteria but also fails to delineate the effects of antibiotic therapy from those of the target bacteria. To overcome this challenge, we attempted to label 11 bacterial isolates from the gut of the eastern tree hole mosquito with three fluorescent markers to facilitate the study of their function in the mosquito without the use of antibiotic therapy. Four bacterial isolates were successfully labeled with at least one of the three fluorescent markers demonstrating that this method can be used for non-destructive studies on bacterial functions within the mosquito.

Technical Abstract: 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.