Preparation of Infectious Wolbachia from a Mosquito Cell Line

Eventual genetic engineering of Wolbachia will require maximizing recovery of infectious bacteria, maintaining Wolbachia in a viable state for efficient manipulation, and reinfection of host cells for propagation and expansion of recombinant progeny. Challenges to manipulating Wolbachia arise from its obligate intracellular lifestyle and inability to divide outside a host cell, requiring modifications of standard bacteriological methods. The Aedes albopictus C7–10 cell line has proven to be a good recipient for the Wolbachia supergroup B strain, wStri, from the planthopper Laodelphax striatellus; the persistently infected C/wStri1 population provides a source of wStri inoculum that can be used systematically to explore conditions that increase yields of infectious material from input Wolbachia and identify conditions conducive to Wolbachia replication. After reintroduction into naive, uninfected C7–10 cells, wStri recovery, relative to the input inoculum, is influenced by diverse conditions, such as the cell cycle arrest that follows treatment of infected host cells with the insect steroid hormone, 20-hydroxyecdysone. Pretreatment of recipient cells with mitomycin C, which cross-links DNA and inhibits host cell replication, can improve recovery from low levels of input Wolbachia. This protocol describes preparation of infectious inoculum from Aedes albopictus C/wStri1 cells and amplification of Wolbachia in mitomycin C-treated, uninfected C7–10 cells, followed by a brief description of conditions used for various small-scale manipulations of Wolbachia in infected cells.