Host cell-free Wolbachia pipientis axenic media formulations and transfection strategies



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Wolbachia pipientis an endosymbiotic found in >55% of insects and is a bacterium of interest for vector control due to its ability to alter host reproduction and block certain pathogens from being transmitted. Host cell-free Wolbachia pipentis was used to determine possible metabolites and conditions that could support future axenic media formulations, and transfection strategies were updated for placement of Wolbachia into target host lines. The data collected will support future Wolbachia axenic media creation, as well as provide a platform for the placement of various Wolbachia strains into target host species.
The metabolic activity of the wAlbB strain of Wolbachia was measured through absorbance on PM1 and PM3 plate types in correlation to differing casamino acid concentrations and pH levels. A casamino concentration of 4mg/mL has a toxic effect on Wolbachia. A 1mg/mL casamino concentration increased metabolic activity in the axenic culture. Altering the pH to 6.5, closer to the internal pH of insect cells, was found to increase Wolbachia metabolism. Using these results, a supplementation experiment was created that compared insect media with altered pH 6.5 and 1m/mL casamino acid. Wolbachia strains wMel and wMelpop were transfected into Aa23 cell lines and maintained through 21 passages. Successful cell line wMel and wMel transfections were aided through the addition of the antifungal amphotericin B and antibiotics streptomycin and penicillin. Multiple transfection procedures completed on the same line over multiple weeks also aided in the establishment of ongoing stable infections. These infections will be used in future metabolic studies.

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Wolbachia, Axenic, Transfection