Proof of concept of the use of paratransgenesis in the Formosan subterranean termite

The Formosan subterranean termite, Coptotermes formosanus, is one of the most economically significant invasive termite species, and therefore, an important target for developing increasingly efficient control methods, preferably without the use of pesticides (Su and Scheffrahn, 1998). In a first experiment leading to proof of concept of paratransgenesis in subterranean termites, Husseneder et al . (2005a) genetically engineered an Escherichia coli laboratory strain with a plasmid that expressed genes for ampicillin resistance and green fluorescent protein (GFP) and fed the genetically modified bacteria to Formosan subterranean termite workers . Workers were screened for the presence of transformed E. coli by culturing their gut bacteria under selective conditions in liquid overnight cultures and on solid agar plates The presence of the GFP reporter gene was confirmed visually by fluorescence, and by PCR amplification of the GFP gene . Although the experiment successfully established that termite workers ingested genetically modified bacteria rapidly through feeding on inoculated filter paper and transferred the bacteria among nest mates, the bacteria did not survive in the termite gut for longer than a week . Because field colonies of subterranean termites can be expansive (Su and Scheffrahn, 1998; Messenger et al , 2005; Husseneder et al , 2005b), a week is probably too short to guarantee spread of genetically modified bacteria and sufficient gene expression levels throughout an entire colony to achieve colony control

To increase the stability of the population of genetically modified microorganisms in the termite gut, Husseneder and Grace (2005) subsequently genetically engineered bacteria that were isolated from the gut of Formosan subterranean termites . Naturally occurring bacteria derived from the termites' own indigenous gut flora should not trigger defensive or immune responses and should be well adapted to the living conditions and selective pressures in the termite gut (Dillon and Dillon, 2004) . Enterobacter cloacae, a strain that was isolated in ample amounts from the gut of Formosan subterranean termite workers (Mannesmann and Piechowski, 1989; Husseneder et al , 2005c), was genetically engineered as the prototype of a shuttle bacterium to express ampicillin resistance markers and GFP Transgenic strains of the genus Enterobacter have previously been used for microbial control in other insect species (Watanabe et al , 2000; Kuzina et al , 2002)

Beard et al. (2002) and Durvasula et al . (2003) listed criteria necessary for a successful strategy using paratransgenesis . These criteria include the ability to (1) isolate and culture symbiotic bacteria from the host insect, (2) genetically engineer these bacteria without loss of fitness, (3) establish methods of reintroduction of genetically modified bacteria into the host, (4) express foreign gene products in the host, and finally, (5) spread the transformed bacteria throughout a target population, and (6) monitor potential environmental contamination

The transgenic E. cloacae shuttle system meets the above criteria (Husseneder and Grace, 2005). For example, Enterobacteriaceae belong to the natural, stable flora of the termites, and are amenable to isolation, culture (Mannesmann and Piechowski, 1989; Husseneder et al , 2005c), and stable transformation with foreign genes without significant loss of viability. A method of delivery of genetically modified bacteria into laboratory colonies has been established: the bacteria were ingested by workers feeding on inoculated filter paper within a few hours, the GFP gene was expressed in the termite gut, and expression persisted in the termite gut for two months Genetically modified bacteria were efficiently transferred throughout the termite colony, among workers and soldiers even when the donor (termites initially fed with transformed bacteria) to recipient (fed with water on filter paper only) ratio was low First assessments of environmental spread of genetically engineered bacteria and possible gene transfer to soil bacteria were conducted in the laboratory by the use of GFP as a traceable marker gene . When termites that contained a population of GFP expressing E. cloacae were kept on soil, genetically modified bacteria were temporarily transferred into soil However, these bacteria did not accumulate in soil but declined over the course of several weeks, which suggests limited survival capability of the genetically modified bacteria among the community of soil bacteria Gene transfer was not observed because all fluorescent bacteria recovered from the soil had the same morphological and biochemical characteristics as the original E. cloacae strain (Husseneder and Grace, 2005) With this study, proof of concept has been established for the use of paratransgenesis to deliver and express foreign genes in termite colonies

The paratransgenic system using GFP bacteria was designed solely as a proof of concept of successful transformation of indigenous symbionts and as a monitoring system for ingestion, long-term survival of genetically modified bacteria in the termite gut, and efficient spread among colony mates To optimize the system for termite control, the following goals have to be achieved Targets and target-specific toxins have to be identified The toxins need to be highly effective against the target, e .g., kill the obligate symbionts that the termite relies on for survival or destroy the termite gut itself, and be slow-acting enough to allow spread throughout the termite colony Genes for these toxins have to be synthesized and the host microorganism has to be genetically engineered to express these target-specific toxins in the termite gut To increase environmental safety, microorganisms have to be identified that are termite specific and thus are unlikely to survive outside the gut However, the bacteria have to be common in the gut of Formosan subterranean termites regardless of their geographic origin and have to be isolated and cultured in the laboratory

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