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. 2021 Sep 28;11:19202. doi: 10.1038/s41598-021-98738-7

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Aedes fluviatilis embryonic cell lines wAflu1 and Aflu2. Embryonic cell line wAflu1 was positive for the Wolbachia-specific gene, WSP, which was absent in Aflu2 cells (A, lane a and c, respectively). Amplification of WSP gene transcripts was still detected in wAflu1 cells after cryopreservation (A, lane b). The WSP fragment amplified by RT–PCR has about 650 pb, as indicated by 100 pb marker (A, lane M). wAflu1 and Aflu2 nuclei were visualized using DAPI staining and laser scanning confocal microscope Zeiss LSM 710, with the presence of Wolbachia being also observable in the cytosol of wAflu1 cells, as indicated by the white arrow in the left panel enlarged from the highlighted quadrant (B). Additionally, nuclei of live cells were stained with Hoechst and visualized by fluorescence microscopy, here merged with phase contrast (ph) images to show overall cell morphology, with granular vesicles in the cytosol (C).