| Summary: | Endosymbiotic intracellular bacteria of the genus <i>Wolbachia</i> are harboured by many species of invertebrates. They display a wide range of developmental, metabolic and nutritional interactions with their hosts and may impact the transmission of arboviruses and protozoan parasites. <i>Wolbachia</i> have occasionally been isolated during insect cell line generation. Here, we report the isolation of two strains of <i>Wolbachia</i>, <i>w</i>Pip and <i>w</i>Pap, during cell line generation from their respective hosts, the mosquito <i>Culex pipiens</i> and the sand fly <i>Phlebotomus papatasi</i>. <i>w</i>Pip was pathogenic for both new <i>C. pipiens</i> cell lines, CPE/LULS50 and CLP/LULS56, requiring tetracycline treatment to rescue the lines. In contrast, <i>w</i>Pap was tolerated by the <i>P. papatasi</i> cell line PPL/LULS49, although tetracycline treatment was applied to generate a <i>Wolbachia</i>-free subline. Both <i>Wolbachia</i> strains were infective for a panel of heterologous insect and tick cell lines, including two novel lines generated from the sand fly <i>Lutzomyia longipalpis</i>, LLE/LULS45 and LLL/LULS52. In all cases, <i>w</i>Pip was more pathogenic for the host cells than <i>w</i>Pap. These newly isolated <i>Wolbachia</i> strains, and the novel mosquito and sand fly cell lines reported here, will add to the resources available for research on host–endosymbiont relationships, as well as on <i>C. pipiens</i>, <i>P. papatasi</i>, <i>L. longipalpis</i> and the pathogens that they transmit.
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