Characterization of Wolbachia Transfection Efficiency by Using Microinjection of Embryonic Cytoplasm and Embryo Homogenate

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Abstract
Wolbachia spp. are intracellular alpha proteobacteria closely related to Rickettsia. The maternally inherited infections occur in a wide range of invertebrates, causing several reproductive abnormalities, including cytoplasmic incompatibility. The artificial transfer of Wolbachia between hosts (transfection) is used both for basic research examining the Wolbachia-host interaction and for applied strategies that use Wolbachia infections to affect harmful insect populations. Commonly employed transfection techniques use embryonic microinjection to transfer Wolbachia-infected embryo cytoplasm or embryo homogenate. Although microinjections of both embryonic cytoplasm and homogenate have been used successfully, their respective transfection efficiencies (rates of establishing stable germ line infections) have not been directly compared. Transfection efficiency may be affected by variation in Wolbachia quantity or quality within the donor embryos and/or the buffer types used in embryo homogenization. Here we have compared Wolbachia bacteria that originate from different embryonic regions for their competencies in establishing stable germ line infections. The following three buffers were compared for their abilities to maintain an appropriate in vitro environment for Wolbachia during homogenization and injection: phosphate-buffered saline, Drosophila Ringer's buffer, and a sucrose-phosphate-glutamate solution (SPG buffer). The results demonstrate that Wolbachia bacteria from both anterior and posterior embryo cytoplasms are competent for establishing infection, although differing survivorships of injected hosts were observed. Buffer comparison shows that embryos homogenized in SPG buffer yielded the highest transfection success. No difference was observed in transfection efficiencies when the posterior cytoplasm transfer and SPG-homogenized embryo techniques were compared. We discuss the results in relation to intra- and interspecific Wolbachia transfection and the future adaptation of the microinjection technique for additional insects.