biostudies-literatureUnknown11(10)Suliman YBacterial endosymbionts are highly prevalent among invertebrate animals, in which they can confer fitness benefits such as pathogen defence and/or act as reproductive manipulators, inducing phenotypes including cytoplasmic incompatibility (CI). For the alpha-proteobacterium <i>Wolbachia</i>, its wide distribution among macroparasites and disease-transmitting arthropods coupled with mutualistic roles, reduction of vector competence and CI has found recent applications in the control of several vector-borne tropical diseases. However, in common with other bacterial endosymbionts, which often lose regulatory elements during genomic erosion, the degree to which <i>Wolbachia</i> can respond to environmental or pharmacological stressors is poorly understood. Here, we apply Cappable-Seq methodology to achieve unprecedented depth and resolution of transcriptional start sites (TSS) in two <i>Wolbachia</i> strains (<i>w</i>MelPop-CLA and <i>w</i>AlbB) that have been used to transinfect mosquitoes for arbovirus control. We exposed <i>Wolbachia</i> in mosquito cell lines to temperature stress (both strains) or antibiotics (<i>w</i>AlbB only) and observed that all classes of TSS (including antisense) exhibited differential regulation, some of which were associated with mobile elements and may control ncRNA expression. Of the three antibiotics used as pharmacological stressors (doxycycline, rifampicin and moxifloxacin), doxycycline had the greatest impact on differential expression from primary TSS. Cappable-Seq also resolved the organization of the bicistronic <i>cifA/cifB</i> operon that is responsible for inducing CI in <i>Wolbachia</i> hosts. The use of Cappable-Seq in this study enabled the resolution of the primary transcriptome of an obligate intracellular bacterium in unparalleled detail. Moreover, this methodology shows great promise for revealing regulation of symbiont functions in whole invertebrates.Microbial genomicshttps://www.ebi.ac.uk/biostudies/studies/S-EPMC12558409biostudies-literatureCappable-Seq reveals the transcriptional landscape of stress responses in the bacterial endosymbiont &lt;i&gt;Wolbachia&lt;/i&gt;.PMC12558409Suliman YSinha ALi ZMakepeace BLEttwiller LDyer PDDarby ACCarlow CKHartley CSfalseCappable-Seq reveals the transcriptional landscape of stress responses in the bacterial endosymbiont &lt;i&gt;Wolbachia&lt;/i&gt;.Bacterial endosymbionts are highly prevalent among invertebrate animals, in which they can confer fitness benefits such as pathogen defence and/or act as reproductive manipulators, inducing phenotypes including cytoplasmic incompatibility (CI). For the alpha-proteobacterium <i>Wolbachia</i>, its wide distribution among macroparasites and disease-transmitting arthropods coupled with mutualistic roles, reduction of vector competence and CI has found recent applications in the control of several vector-borne tropical diseases. However, in common with other bacterial endosymbionts, which often lose regulatory elements during genomic erosion, the degree to which <i>Wolbachia</i> can respond to environmental or pharmacological stressors is poorly understood. Here, we apply Cappable-Seq methodology to achieve unprecedented depth and resolution of transcriptional start sites (TSS) in two <i>Wolbachia</i> strains (<i>w</i>MelPop-CLA and <i>w</i>AlbB) that have been used to transinfect mosquitoes for arbovirus control. We exposed <i>Wolbachia</i> in mosquito cell lines to temperature stress (both strains) or antibiotics (<i>w</i>AlbB only) and observed that all classes of TSS (including antisense) exhibited differential regulation, some of which were associated with mobile elements and may control ncRNA expression. Of the three antibiotics used as pharmacological stressors (doxycycline, rifampicin and moxifloxacin), doxycycline had the greatest impact on differential expression from primary TSS. Cappable-Seq also resolved the organization of the bicistronic <i>cifA/cifB</i> operon that is responsible for inducing CI in <i>Wolbachia</i> hosts. The use of Cappable-Seq in this study enabled the resolution of the primary transcriptome of an obligate intracellular bacterium in unparalleled detail. Moreover, this methodology shows great promise for revealing regulation of symbiont functions in whole invertebrates.2025-01-01T00:00:00Z2025 Oct2026-06-05T21:53:15.311Z2026-05-22T03:16:12.565ZS-EPMC125584094114385810.1099/mgen.0.001542