Depletion of host cell riboflavin reduces Wolbachia levels in cultured mosquito cells.

Wolbachia is an obligate intracellular alphaproteobacterium that occurs in arthropod and nematode hosts. Wolbachia presumably provides a fitness benefit to its hosts, but the basis for its retention and spread in host populations remains unclear. Wolbachia genomes retain biosynthetic pathways for some vitamins, and the possibility that these vitamins benefit host cells provides a potential means of selecting for Wolbachia-infected cell lines. To explore whether riboflavin produced by Wolbachia is available to its host cell, we established that growth of uninfected C7-10 mosquito cells decreases in riboflavin-depleted culture medium. A well-studied inhibitor of riboflavin uptake, lumiflavin, further inhibits growth of uninfected C7-10 cells with an LC50 of approximately 12 µg/ml. Growth of C/wStr1 mosquito cells, infected with Wolbachia from the planthopper, Laodelphax striatellus, was enhanced in medium containing low levels of lumiflavin, but Wolbachia levels decreased. Lumiflavin-enhanced growth thus resembled the improved growth that accompanies treatment with antibiotics that deplete Wolbachia, rather than a metabolic advantage provided by the Wolbachia infection. We used the polymerase chain reaction to validate the decrease in Wolbachia abundance and evaluated our results in the context of a proteomic analysis in which we detected nearly 800 wStr proteins. Our data indicate that Wolbachia converts riboflavin to FMN and FAD for its own metabolic needs, and does not provide a source of riboflavin for its host cell.

The oxidizing agent, paraquat, is more toxic to Wolbachia than to mosquito host cells.

Cultured cells provide an important in vitro system for examining metabolic interactions between the intracellular bacterium, Wolbachia pipientis, and its insect hosts. To test whether Wolbachia-associated changes in antioxidant activities could provide a tool to select for infected cells, we tested the effects of paraquat (PQ) on Aedes albopictus mosquito cells. Like mammalian cells, mosquito cells tolerate PQ over a wide range of concentrations, and for considerable lengths of time, depending on cell density at the time of treatment. When mosquito cells were plated at low density and allowed to grow in the presence of PQ, we measured an LC50 of approximately 1-2 µM. Unexpectedly, cells persistently infected with Wolbachia strain wStr, from the planthopper Laodelphax striatellus, grew to higher densities in the presence of 1.5 µM PQ than in its absence. This effect of PQ was similar to the improved growth of host cells that occurs in the presence of antibiotics that suppress the Wolbachia infection. A more detailed examination of growth and metabolic sensitivity indicated that wStr is about 10-fold more sensitive to PQ than the mosquito host cells. Microscopic examination confirmed that Wolbachia levels were reduced in PQ-treated cells, and DNA estimates based on the polymerase chain reaction (PCR) indicated that Wolbachia abundance decreased by approximately 100-fold over a 10-d period. Although Wolbachia genomes encode superoxide dismutase, inspection of annotated genomes indicates that they lack several genes encoding products that ameliorate oxidative damage, including catalase, which converts the PQ byproduct, hydrogen peroxide, to molecular oxygen and water. We suggest that loss of multiple genes that participate in repair of oxidative damage accounts for increased sensitivity of Wolbachia to PQ, relative to its host cells.