Mitotically inactivated mosquito cells support robust Wolbachia infection and replication.

Wolbachia is an obligate intracellular bacterium that infects many species of insects, and has been of particular interest in recent efforts to reduce disease transmission by mosquitoes. Two aspects of Wolbachia biology underlie its applications for insect control: first, the bacterium behaves as a natural gene drive agent and, second, when introduced into mosquitoes that do not harbor Wolbachia in nature, infection reduces survival of pathogens. These properties support efforts to explore the basic biology of Wolbachia in insect cell lines, which can produce sufficient infectious material for microbiological studies and microinjection into novel hosts. When introduced into naïve C7-10 Aedes albopictus mosquito cells, the yield of Wolbachia strain wStri improves, roughly in proportion to the size of the inoculum, as exponential growth of the host cell ceases. Wolbachia yields also increase when persistently infected C/wStri1 cells or naive, newly infected cells are treated with 20-hydroxyecdysone (20E), which inhibits growth in the G1 phase of the cell cycle. These observations suggest that Wolbachia infection and replication are independent of exponential growth and mitosis of host cells. To explore yields of infectious bacteria in cells arrested prior to infection, I tested host cells pre-treated with mitomycin C, an agent that crosslinks DNA and prevents cell division that is used to produce "feeder layers" with mammalian cells. Yields of wStri per plate increased by about 50-fold relative to exponentially growing cells, and the multiplicity of infection necessary for a robust infection was reduced to a single bacterium per cell. These results suggest that Wolbachia infection and replication are supported by mitotically arrested cells and provide new insights into biological processes that influence maintenance of a widespread obligate intracellular bacterium.