Genetic engineering of a termite specific bacterium using a Tn7 transposon vector for paratransgenesis based termite control

The gut of the Formosan subterranean termite Coptotermes formosanus Shiraki harbors a complex community of bacteria and protozoa. The gut protozoa help termites to digest cellulose, and in their absence, termites die of starvation. We previously showed that the lytic peptide Hecate coupled to a ligand that binds to protozoa, kills protozoa and ultimately termites. In this study we isolated 15 bacteria species from the termite gut with the goal to use them as Trojan Horses for expression of ligand-Hecate in the termite colony. These bacteria were tested for their tolerance against ligand-Hecate. Bacteria were tolerant to ≥ 25x higher concentrations of ligand-Hecate than required to kill the gut protozoa. Trabulsiella odontotermitis, a termite specific bacterium, was genetically engineered with a plasmid expressing GFP and fed to workers. The experiment confirmed ingestion of engineered T. odontotermitis and its survival in the gut for ≥ 48 hrs. Next, the genes need to be inserted into the T. odontotermitis chromosome to ensure long term stability of gene expression. A Tn7 transposon based vector capable of constitutively expressing GFP was constructed and used to insert the gene cassette into the chromosome. Transformed T. odontotermitis expressing GFP from the chromosome will be used in feeding assays to assess long term stability and transfer among colony mates. Next, the Tn7 transposon vector will be used to engineer T. odontotermitis to express ligand-Hecate. Ultimately, we envision the application of T. odontotermitis expressing ligand-Hecate as a Trojan horse that can be formulated within baits for termite control.