Localization analysis of symbionts
We fixed H. acanthopus tissues by incubation in 4% paraformaldehyde solution at 4°C for 39 h. Subsequently, insects were transferred and kept at 4°C in Carnoy’s solution for 27 h, 2% hydrogen peroxide ethanol solution for 3 days and 6% hydrogen peroxide ethanol solution for 10 days to quench tissue autofluorescence. We then washed specimens with 400 µl of hybridization buffer (900 mM NaCl, 20 mM Tris-HCl pH 7.4, 0.01% sodium dodecyl sulfate, SDS, and 30% formamide) at 46°C for 10 min, pre-hybridized with 200 µl of hybridization buffer at 46°C for 1 h, and hybridized at 46°C for 3 h with 400 µl of hybridization buffer containing probes. All fluorescent probes were obtained from Sigma-Aldrich (St. Louis, Missouri, USA) and used at a concentration of 2.5 ng μl-1 for Cyanine 5 (Cy5)-labelled EUB338 (5’- GCTGCCTCCCGTAGGAGT -3’), targeting all bacteria (Amann, Krumholz, & Stahl, 1990) and 6-carboxyfluorescein (6-Fam)-labelled beta-572 (5’- TTAACCGTCTGCGCTCGCTT -3’), targeting the family Neisseriaceae (Martinson, Moy, & Moran, 2012). We pre-evaluated the required stringency of the hybridization conditions in silicousing mathFISH (Yilmaz, Parnerkar, & Noguera, 2011). Following hybridization, we washed specimens twice with 400 µl of pre-warmed wash buffer (20 mM Tris-HCl, 5 mM EDTA, 0.01% SDS and 112 mM NaCl) at 48°C for 10 min. All incubations were carried out with ongoing shaking at 300 rpm. We then placed lice on microscope slides, incubated them in ~50 μl of 4’,6-diamidino-2-phenylindole (DAPI) solution (1 ng μl-1) in the dark for 10 min, and mounted slides in Mowiol anti-fading medium (Kuraray Europe GmbH, Tokio, Japan). We captured the fluorescent signals with a laser scanning confocal microscope Olympus FV3000 (Olympus, Tokio, Japan). We acquired at least three confocal stacks (up to 30 scans per optical slice) at 100x, 400x and 630x magnifications, a color depth of 24 bit and a resolution from 1 to 2 μm per pixel (depending on the fluorochrome) by investigating multiple regions from each of the three replicate specimens. Resultant images were processed with the ImageJ distribution Fiji (Schindelin et al., 2012; Schneider, Rasband, & Eliceiri, 2012)