Analysis of Bacterial Communities Associated with Insect Biological Control Agents using Molecular Techniques

Investigations of the diversity of bacterial communities associated with fieldcollected specimens of two insect biological control agents of spotted knapweed, Agapeta zoegana and Cyphocleonus achates, were made using molecular methods. The objective was to assess the bacterial communities of each insect to evaluate the potential compatibility of plant pathogenic fungi as a supplement to insect diets for mass rearing of the two species. Synergistic interactions of plant pathogenic fungi with root-attacking insects have been extensively correlated with reductions in stand density of rangeland weeds. This synergism has been noted both in foreign and domestic stands of leafy spurge (Euphorbia esula) and spotted knapweed (Centaurea maculosa). The bacterial flora of larvae of the biological control agent Aphthona nigriscutis of Euphorbia esula was also investigated as an indication of possible factors that may affect their establishment, survival and capacity to impact the weed. After DNA extraction of whole larvae, the V6 to V8 region of 16S rDNA was PCR-amplified. Community profiles were obtained using denaturing gradient gel electrophoresis (DGGE) and isolated bands were excised, reamplified and sequenced for phylogenetic comparisons. Phylogenetic analysis indicated that some bands represented microorganisms causing ice nucleation, antagonism toward soilborne plant pathogens and symbionts associated with mating incompatibility. The results suggest that these insect biological control agents maintain diverse microbial communities, an important consideration in formulating artificial diets that may contain antibiotics or other preservatives. Furthermore, the findings imply that microbial communities associated with insects influence the establishment, and impact of these insects.