Dynamics and Fitness Cost of Genetically Engineered Entrobacter cloacae Expressing Defensin for Paratransgenesis in Phlebotomus papatasi

Background: Enterobacter cloacae subsp. dissolvens bacterium is a known commensal of the gut microflora of Phlebotomus papatasi, the main vector for zoonotic cutaneous Leishmaniasis, and nominated for paratransgenesis in sand flies. In this study, we evaluated dynamics and fitness costs of engineered E. cloacae for its potential to serve as a 'Trojan Horse' in P. papatasi. Methods: The engineered strain of E. cloacae transformed with a constantly active expressed red fluorescent protein plus defensin (EC-DR) plasmid and was fed to sand fly colonies via larval food to larvae. A wild type the bacterium (EC-WT) and intact food were used as controls. Fitness characters as well as dynamics of the EC-DR at various development stages of sand fly larvae were tested by plating homogenized specimens and counting fluorescent expressing colonies on the Tet-BHI agar medium. Results: Enterobacter cloacaeDR producing red fluorescent protein could be isolated from the larvae gut after 36 days when the bacteria were added once in larval pots. The EC-DR with multiple applications had no negative effect on emergence time of instar II larvae, pupae, and adults but increased slightly mortality rate of P. papatasi larvae. The experiment also confirmed lack or weak trans-stadial transmission of E. cloacae DR in P. papatasi. It has minimal fitness cost on P. papatasifeeding behavior and survival. Conclusion: Results of this study showed that E. cloacae DR is suitable for paratransgenesis of P. papatasi at only adult stage because it did not transmit transstadially.