Dfc450 camera

Half-gravid females were sampled in November 2016 (Table S1) in forest sites where we previously found specimens belonging to the unspecified taxon. Females were collected by HLC and fed to complete their blood-meal. Mosquitoes were allowed to develop follicles for 25 h at field temperature. Then, ovaries were dissected and stored in Carnoy’s fixative solution (100% ethanol: glacial acetic acid, 3:1 by volume). At the CIRMF, ovaries were squashed in a drop of 50% propionic acid to obtain the polytene chromosomes⁶² . The banding patterns of polytene chromosomes were examined using a Leica DM2000 microscope equipped with a Leica DFC 450 camera system (Leica Microsystems GmbH, Wetzlar, Germany). Chromosomal arms and inversions were recorded and scored according to the An. gambiae chromosome map^{63 (link)}.

ANOVA (one-way analysis of variance) was applied followed by Dunnett’s multiple comparison tests for statistical calculations. A burst of cellular images was taken by Leica DFC450 camera (Leica Microsystems GmbH, Wetzlar, Germany) at constant exposure time, gain, color saturation and gamma adjustment. CTCF (corrected total cellular fluorescence) was calculated in the ImageJ software (NIH, Bethesda, MD, USA). Mean cellular fluorescence got subtracted by mean background (non-cellular) fluorescence. Specific statistical statements are given in respective figure legends.

Subsequent to TTFields treatment, cells were washed three times with PBS and fixed in pre-chilled methanol for 20 min at −20 °C. Next, cells were washed again and blocked in 5% donkey serum (DS, Abcam, Cambridge, UK) diluted in PBS for 1 h at room temperature. The cells were afterwards incubated with the primary antibodies mouse anti-claudin-5 conjugated to Alexa Fluor 488 (1:500, Thermo Scientific, Cat. No. 352588), mouse anti-zonula occludens-1 conjugated to Alexa Fluor 488 (1:500, Thermo Scientific, Cat. No. 339188), and rabbit anti-PECAM-1 (1:500, Novus Biologicals, Centennial, CO, USA, Cat. No. NB100-2284) in 1% bovine serum albumin (BSA)/PBS with 5% DS overnight at 4 °C. On the following day, the cells probed with rabbit anti-PECAM-1 were probed with the secondary antibody anti-rabbit Alexa Fluor 555 (1:400 in 1% BSA/PBS with 5% DS, Invitrogen, Cat. No. A-21429) for 1 h at room temperature. In addition, the cells were then washed three times with PBS. Finally, the cover slips were mounted on glass microscope slides using Fluoroshield mounting medium with DAPI (Abcam), allowed to dry, and subsequently viewed under the microscope. Five representative fields of view per slide were photographed with the LEICA DMI 3000 B microscope, LEICA DFC 450 camera, and LAS V4.5 software (all Leica Microsystems, Wetzlar, Germany) with standardized settings at 40× magnification.