Bacterial diversity in the gut of the Formosan subterranean termite

A prototype gene-shuttle was assembled in yeast and used in termite feeding experiments; death of the protozoa in the termite gut provided proof of concept for the use of paratrans-genesis in termite control The ultimate goal for environmentally safe field applications, however, is to use novel bacteria that are exclusively found in the termite gut as shuttles and expression systems Termite-specific bacteria species would not be able to survive for a prolonged time outside the termite gut and thus reduce the potential environmental impact, including the persistence of the transgenic bacteria in soil and possible gene transfer between bacterial strains

Bacterial diversity has been studied in several subterranean termite species, for example, Reticulitermes speratus (Ohkuma and Kudo, 1996; Hongoh et al ., 2003), R. flavipes (Fisher et al ., 2007), and C. formosanus (Shinzato et al ., 2005; Husseneder et al ., 2005c) using culture-independent methods such as sequencing or restriction fragment length analysis of 16S rRNA genes Shinzato et al (2005) found 49 bacteria species in the guts of Formosan subterranean termites in Japan, including 39 novel species . Most of the remaining species belonged to the phyla Bacteroidetes, Firmicutes, and Spirochaetes. We are currently compiling an inventory of bacterial species found in Formosan subterranean termite colonies from its introduced range, i e , Louisiana, Hawaii (Husseneder et al , 2005c) and Japan (Shinzato et al , 2005) and from its native range, i e , China (Ho and Husseneder, 2007) to identify species that are obligate and common across geographical regions To date, over 220 bacteria species from ten bacterial phyla were identified; over 80% were novel species, yet the majority clustered closely with bacteria lineages found only in subterranean termites and not in the environment (Hongoh et al , 2003; Shinzato et al , 2005) The diversity of bacteria in the termite gut provides a wide range of "raw material" for genetic transformation and use as a Trojan horse The fact that most of these bacteria are specific to termites and are not likely to survive outside of the termite gut would increase environmental safety

The ideal host bacterium for a paratransgenic approach would be a novel species that is culturable, but unlikely to survive in the environment for a prolonged period of time, and common in all colonies of the Formosan termite regardless of geographical region, but specific to this termite species A novel Bacteroides species, which was dominant in the bacterial inventory of Formosan termites introduced to Japan (Shinzato et al , 2005), also was dominant in colonies in Louisiana (Husseneder et al , 2005c) and present in lower propor tions in Chinese colonies (Ho and Husseneder, 2007) However, this species has not been cultured yet . The most prominent culturable species that was found in Formosan subterranean termite colonies from Japan, Louisiana, and China was Pilibacter termitis, a novel species, which had been previously isolated from colonies in Hawaii (Husseneder et al , 2005c) and was described by Higashiguchi et al. (2006). Pilibacter termitis is a Gram-positive, lactic acid bacterium Gram-positive bacteria are considered less susceptible to the membrane disruptive activity of lytic peptides than Gram-negative bacteria (Javadpour et al ., 1996) . Thus, Pilibacter termitis is a promising candidate for genetic transformation to become a bacteria shuttle A limitation to the use of termite-specific novel bacteria species, such as P. termitis, is that protocols for genetic manipulation of this species do not exist in the literature Transformation protocols are currently being developed based on methods used on phylogenetically similar bacteria . Even if the genetic transformation of P. termitis should not be successful, dozens of less frequent bacterial strains that were previously cultured from the diverse gut flora of the Formosan subterranean termite could be genetically engineered These cultured bacteria range from novel species to common Enterobacteria-ceae (Mannesmann and Piechowski, 1989; Osbrink et al ., 2001; Husseneder et al ., 2005c) .

Alternatively, yeast remains a promising candidate host for the paratransgenesis system, especially with safety precautions, such as the addition of a ligand targeting the lytic peptide to the surface of the protozoa or the gut wall or with the activation of the secreted lytic peptide by termite-specific gut enzymes . The use of yeast as the Trojan horse has the advantage that it could be incorporated in existing bait systems in freeze-dried form, resulting in a long shelf life and easy application to termite colonies

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