Jason M. Meyer, hessianfly@yahoo.com and Marjorie A. Hoy, mahoy@ifas.ufl.edu. University of Florida, Department of Entomology & Nematology, Gainesville, FL
Molecular surveys of insect symbionts are increasing due to the importance of internal microbes in the biology of their host. Presently, no consistent method has been reported to eliminate surface microbes from insects in these surveys. This research is needed because an overestimation of the total microbial fauna can arise when Polymerase Chain Reaction (PCR) assays amplify DNA isolated from surface microbes mixed with insect and endosymbiont DNA. Experimental costs increase when DNA amplified from external microbes is cloned, sequenced and analyzed. The microbial community present on the surface of the Asian citrus psyllid, Diaphorina citri Kuwayama (Homoptera: Psyllidae), was examined by culturing experiments using nutrient agar plates. DNA was isolated from microbes grown in culture and used as a template to PCR-amplify ribosomal RNA genes for sequencing and identification. A series of surface decontamination washes was administered to adult psyllids, including combinations of bleach, Tween 20, DNase, DNA lysis buffer and sterile water. The efficacy of each method was tested by PCR amplification of microbial sequences previously identified in the culturing experiments.
Species 1: Hemiptera Psyllidae
Diaphorina citri (Asian citrus psyllid)
Keywords: Endosymbionts, Decontamination