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

Bacteria from the hindguts 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 hindguts of D. albohirtum larvae were from the Proteobacteria, Firmicutes, and Actinobacteria phyla and matched closely with taxa from intestinal and rhizosphere environments. However, these isolates were not the most common gut-associated bacteria identified in denaturing gradient gel electrophoresis (DGGE) hindgut profiles. Subsequently, eight 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 postconsumption. 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 transposon system, a kanamycin resistance gene was inserted into 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 I X 106 transgenic Da-11 cells/ml in the hindguts of larvae at 9 days postconsumption. These populations were maintained in the hindgut for at least another 12 days. The successful isolation, genetic transformation, and establishment of transgenic Da-11 cells in the hindguts of D. albohirtum larvae fulfill fundamental requirements for the future development of a paratransgenic approach to control scarab pests of sugarcane.