Aseptic rearing procedure for the stinkbug Plautia stali (Hemiptera: Pentatomidae) by sterilizing food-derived bacterial contaminants

The stinkbug Plautia stali Scott is a notorious agricultural pest whose posterior midgut hosts specific bacteria essential for its growth and survival, highlighted as an experimental model for symbiosis studies. Some symbiotic bacteria of P. stali are cultivable, found free-living in and acquired from the environment, and, furthermore, some free-living environmental bacteria are potentially capable of establishing symbiotic association with P. stali. In this context, it is expected that such environmental bacteria may occasionally contaminate and infect the experimental insects maintained in the laboratory, which could potentially affect the functional analyses of the symbiosis. Here we report that such contamination events do occur under a laboratory rearing conditions for P. stali. When symbiont-deprived newborn nymphs from surface-sterilized eggs were reared in sterilized plastic containers with autoclaved water, most of them died as nymphs presumably as a result of aposymbiosis, but only a small fraction could attain adulthood and the adult insects were all infected with γ-proteobacteria allied to Pantoea and Enterobacter. A variety of bacteria, mainly Bacillus and also Pantoea and Enterobacter, were detected from peanuts and soybeans provided as food for P. stali. Autoclaving of peanuts and soybeans eradicated these bacteria but negatively affected the host survival, whereas ethanol sterilization of peanuts and soybeans removed Pantoea and Enterobacter, but not Bacillus, without negative effects on the host survival. On the basis of these results, we established a practical procedure for aseptic rearing of P. stali, which will enable reliable and strict analyses of host–symbiont interactions in the model symbiotic system.