Planapo z objective

Fluorescence microscopy of GFP signals was optimized for live cell and detected in roots during 10 days every 24 h after transfer the plants on N-depleted medium (Svietlova et al., 2023 (link)). Images were acquired using Zeiss AXIO Zoom.V16 (ZEISS, Germany, Oberkochen) equipped with 0.5× PlanApoZ Objective (ZEISS, Germany, Oberkochen), an HXP 120 mercury vapor lamp and a filter set 38 HE (excitation filter BP 450-490nm, FT 495nm, emission filter BP 500-550nm) for the visualization of GFP. Signal intensities after treatment were measured using Fiji ImageJ-2.9.0 Analysis Software. Images were converted to 8-bit and processed using Fiji’s “analyze particles” plugin. The average fluorescence intensity was measured in the cells of the apical lateral roots. For the measurement, ten randomly selected fluorescent points in the form of a square of four pixels for each plant were used. Fluorescence images were captured using a TOMOCUBE HT-X1 (Tomocube Inc., Republic of Korea) on 6th day of N starvation. HT-X1 model includes a 470 nm LED source, which was used to acquire 3D fluorescence images of GFP.

To build the posterior lobe atlas, 20 males and 20 females from different species were forced to mate in a vial with standard fly food and the posterior lobes of the resulting male offspring (at the age of 7 days after eclosion) were dissected. All male flies including the pure and hybrids were 7-day-old virgins. During dissection, the flies were anesthetizing by CO₂. The posterior lobe was separated from male genital by using a scalpel and a needle. Posterior lobes were put on microscope slides (Paul Marienfeld GmbH & Co. KG, Germany), immersed by methyl salicylate (Sigma-Aldrich, Germany), and covered by a coverslip (Carl Roth GmbH & Co. KG, Germany). On each slide, 15 posterior lobes from different individuals were placed. Slides were photographed using an AXIO Zoom V.16 (ZEISS, Germany, Oberkochen) with a 1.25× PlanApo Z objective (ZEISS, Germany, Oberkochen).

Dispatched flies were mounted and views of the ovipositor (180x) were acquired as focal stacks on an AXIO Zoom V.16 (ZEISS, Germany, Oberkochen) with a 0.5x PlanApo Z objective (ZEISS, Germany, Oberkochen). The resulting stacks were compiled to extended focus images in Helicon Focus 6 (Helicon Soft, Dominica) using the pyramid method. Here we provided images of the serrated ovipositors of close relatives to D. suzukii in order to highlight the physical deviations in egg-laying potential (Figure S1), which has been described previously (Atallah et al., 2014 ). We also documented the differences in male wing pigmentation (32x), as D. suzukii and D. subpulchrella can be difficult to distinguish (Figure S7). Moreover, D. subpulchrella were shown recently in the most up to date phylogenetic analyses of this Drosophila clade to be the closest relatives to D. suzukii, as opposed to D. biarmipes (Dekker et al., 2015 ; Hickner et al., 2016 ; Keesey et al., 2019a (link); Ramasamy et al., 2016 (link)). Images were also compiled of dissected heads (128x) to illustrate the mounting preparations for sensillum counts (Figures 2A–2D).