Las version 4

We followed the methods of Lee et al. [18 (link)]. Briefly, cells were incubated with fluorescein diacetate (FDA, 5 μg/mL) (Sigma-Aldrich) and PI (10 μg/mL) (Sigma-Aldrich) in the dark, to stain live and dead cells, respectively. The FDA, a cell-permeable esterase substrate, serves as an indicator for viable cells by assessing enzymatic activity and cell-membrane integrity, while PI passes through damaged areas of dead or dying cell membranes into the nucleus and binds to DNA. Cells were imaged using a Leica EL6000 fluorescence microscope and LAS version 4.3 software (Leica Microsystems GmbH).

The abdomen of each adult female P. jenynsii was cut to make a slit at the midline using ophthalmological microscissors in PBS. Then, the insects were fixed in PBS containing 4% paraformaldehyde at 4°C overnight. After several washes with PBS, the specimens were subjected to wFISH as previously described (Koga et al., 2009 (link)). The specimens were hybridized with Al555-EUB338 (5'-Alexa Fluor 555- GCT GCC TCC CGT AGG AGT-3') targeting bacterial 16S ribosomal RNA. After overnight hybridization at room temperature, the specimens were washed with PBT (PBS containing 0.1% Tween-20) containing 165 nM Alexa Fluor 488 phalloidin and 2 μg/mL 4',6-diamidino-2-phenylindole (DAPI) for 30 min at room temperature and subsequently washed with PBT three times. The hybridized specimens were dissected and pictured in PBS under a fluorescence dissection microscope M165 controlled by LAS version 4.13.0 (Leica). The bacteriomes on the abdominal cuticle were mounted in PBS-50% glycerol and observed with a laser scanning microscope LSM700 controlled with Zen 2011 version 7.0 (Zeiss). Digital images were manually manipulated using Affinity Photo version 2.1.1 (Affinity).