Ic80 hd

Colony area estimated by visualizing live colonies with a Leica M80 stereoscope at 10X magnification and acquiring color images in JPEG format with a Leica IC80HD camera. Images were imported into ImageJ as implemented in Fiji [55 (link)]. This was converted into a binary black and white image by adjusting the threshold level until all pixels within the colony perimeter were black and the background remained white. The “Analyzed Particles” command was then used to compute the area of the colony. The scale was determined by imaging a ruler at the same magnification. Statistical analysis was performed in GraphPad Prism.

Xfp and Xff growth was assessed adding CH-nanoFos-Al to PD2 medium (final concentration of 100 µg/mL, 10 µg/mL and 1 µg/mL) following Bleve et al. 2018. Xfp strain De Donno and Xff strain Temecula1 were grown in PD2 broth for 7 days at 28 °C and 100 rpm. Then, 10 μL of decimal dilutions from 10⁵ to 10¹ CFU mL⁻¹ of each inoculum were applied to the PD2 agar plates supplemented or not with CH-nanoFos-Al. The agar plates were incubated at 28 °C for 8 days for Xff and 20 days for Xfp. Four replications were performed for each dilution and each experiment was carried out two times (n = 8). All images were captured by using a stereo microscope (Wild Heerbrugg, Heerbrugg, Switzerland) connected to a digital camera (Leica IC80 HD) in the same conditions of bright field illumination. CFU counting and total area analysis was optically quantified, for each image, after correcting image contrast and brightness at the same conditions, by using ImageJ software 1.52v.

Embryos between 12 to 48 hpf were categorized into dorsoventral patterning phenotypes (Figure 1g). All images of embryos were photographed in E3 media with a Leica IC80HD. Injection results and controls from multiple experiments were then pooled. Phenotype pictures were corrected with background subtraction using ImageJ, and white balanced using Adobe Photoshop. Embryo bubble graphs were generated by pooling total numbers of embryos within each phenotypic category (C5-V5, Figure 1g) in each condition. These numbers were converted into percent of total embryos to generate the bubble graphs using excel bubble graph. (Figure 1—source data 1, Figure 1—figure supplement 2—source data 1, Figure 3—source data 1, Figure 5—source data 1, Figure 5—figure supplement 2—source data 1, Figure 6—source data 1, Figure 7—source data 1).

Guts from control and CapaR knockdown flies were dissected in artificial hemolymph (AHL) with minimal disruption of attached tissues and without removing the head. Individual exposed guts were next pinned onto a Sylgard-lined petri dish, with fine tungsten pins through the proboscis and a small piece of cuticle attached to the end of each gut and bathed in 100 µl of AHL. Following a 5-minutes acclimation period, each gut was then recorded with a Leica IC80 HD camera mounted on a Leica M50 stereomicroscope for 3-minutes, before the solution was exchanged with 100 µl of AHL and recorded for another 3-minutes. This was done to correct for potential artefacts on gut contraction frequency associated with the exchange of solutions. Next, the AHL solution was replaced with 100 µl of 10⁻⁷ M of either Capa-1 or Capa-2 peptide in AHL, and tissues recorded for an additional 2 × 3-min. Contractions of the entire gut were visually counted post image acquisition, and the number of contractions was normalized to the number of contractions observed in the initial AHL solution with n = 6–8 guts counted for each genotype.

We use the term variable for any categorical (binary or multistate) or quantitative (continuous, discrete) attribute. Floral variables were examined on fresh samples under a Leica M60 stereomicroscope at 6.4× to 50× magnification. High-definition microphotographs were obtained using a Leica IC80HD digital camera (e.g., Figure 6), and measurements were performed on the images using the application LAS ver. 3.8. for Leica Instruments. Measurements of the whole corolla, sepal, and peduncle (after gently straightening it) were gathered on fresh material using a digital calliper with 0.01 mm precision. Measurements of leaves underlying peduncles were performed on material removed from fresh plants, then pressed and dried. Data were gathered for 21 quantitative and 11 categorical variables previously considered as systematically crucial [7 (link),14 ,16 ,53 ,54 (link),62 ,73 ]. (Figure 6, Table 6). According to the literature, ten ratios were derived (Table 6), presuming to reflect among-species differences better than the individual variables [9 (link)]. The floral and vegetative datasets were treated separately. Neither the fruiting peduncle length nor the bracts position on the peduncle was individually computed. The latter, more reliable when computed in the fruit-bearing peduncle than in the floral [7 (link),54 (link)], was unavailable given the adopted sampling protocol.