Assessment of gut-bacteria for a paratransgenic approach to control Dermolepida albohirtum larvae

School of Integrative Biology, The University of Queensland, St. Lucia, Qld 4072, Australia; BSES Limited, PO Box 86, Indooroopilly, Qld 4068, Australia; Australian Institute for Bioengineering and Nanotechnology, c/o - BSES Limited, PO Box 86, Indooroopilly, Queensland 4068, Australia

^* To whom correspondence should be addressed. Email: scott.oneill{at}uq.edu.au.

	Abstract

Bacteria from the hindgut of Dermolepida albohirtum larvae were assessed for their potential to be used in paratransgenic strategies that target scarab pests of sugarcane. Bacteria isolated in pure culture from the hindgut of D. albohirtum larvae were from the Proteobacteria, Firmicutes and Actinobacteria phyla, and matched closely to taxa from intestinal and rhizosphere environments. However these isolates were not the most common gut-associated bacteria identified in DGGE hindgut profiles. Subsequently, 8 species of gut-bacteria were fed to larvae and RNA-based DGGE analysis of 16S rRNA was used to detect the persistence of these isolates in the hindgut environment. One of these isolates (Da-11) remained metabolically active in the hindgut for 19 days post-consumption. Da-11 most likely forms a new genus within the Burkholderiales order, along with taxa independently identified from larvae of the European scarab pest, Melolontha melolontha. Using the EZ::Tn5^TM transposon system, a kanamycin resistance gene was inserted onto the chromosome of Da-11, thus establishing a stable transformation technique for this species. A second feeding trial that included inoculating approximately 400 transgenic Da-11 cells onto a food source resulted in a density of 1 x 10⁶ transgenic Da-11 cells/ml in the hindgut of larvae 9 days post-consumption. These populations were maintained in the hindgut for at least a further 12 days. The successful isolation, genetic transformation and establishment of transgenic Da-11 cells in the hindgut of D. albohirtum larvae fulfils fundamental requirements for the future development of a paratransgenic approach to control scarab pests of sugarcane.