Dynamics of Transgenic Enterobacter cloacae Expressing Green Fluorescent Protein-Defensin (GFP-D) in Anopheles stephensi under Laboratory Condition

Abstract

Background: Enterobacter cloacae bacterium is a known symbiont of the most Anopheles gut microflora and nominated as a good candidate for paratransgenic control of malaria. However, the population dynamics of this bacterium with­in An. stephensi and its introduction methods to the mosquitoes have not yet been explored. Methods: Enterobacter cloacae subsp. dissolvens expressing green fluorescent protein and defensin (GFP-D) was used to study transstadial transmission and the course of time, larval habitat, sugar, and blood meal on dynamics of the bacterium in the mosquito life stages in the laboratory condition. The bacterial quantities were measured by plating samples and counting GFP expressing colonies on the Tet-BHI agar medium. Results: The E. cloacae population remained stable in sugar bait at least for eleven days whereas it was lowered in the insectary larval habitat where the bacteria inadequately recycled. The bacterium was weakly transmitted transstadi­ally from larval to adult stage. The bacterial populations increased smoothly and then dramatically in the guts of An. stephensi following sugar and blood meal respectively followed by a gradual reduction over the time. Conclusion: Enterobacter cloacae was highly stable in sugar bait and increased tremendously in the gut of female adult An. stephensi within 24h post blood meal. Sugar bait stations can be used for introduction of the transgenic bacteria in a paratransgenic approach. It is recommended to evaluate the attraction of sugar bait in combination with attractive kairomones as well as its stability and survival rate in the semi-field or field conditions.

Keywords

• Bacterial dynamic
• Enterobacter cloacae
• Anopheles stephensi
• Paratransgenesis