Porous polycarbonate membrane

A total of 4×10⁴ cells in 200 μl serum-free medium were added to the upper chamber of 24-well culture inserts with a porous polycarbonate membrane that was (8.0 μm, Millipore, Billerica, MA, USA) pre-coated (for invasion) with 30 μl Matrigel (BD Biosciences, San Jose, CA, USA) or not pre-coated (for migration) for 3 hours, and complete media was placed in the lower chamber. The plates were incubated for 24 hours at 37°C in 5% CO₂. After removing the cells from the upper surface of the membrane with a cotton swab, cells on the lower surface were stained with crystal violet sulfate and counted under a microscope (Olympus, Tokyo, Japan).

During the summer (June) of 2018 to spring (March) of 2020, 192 water samples in the experimental area and the control area of the artificial structures were collected in four different depths (0, 2, 4 and 6 m) in 8 quarters (Fig. 1). Each of the two groups had 5 randomly repeated sampling points which include AS1-AS5 and CW1-CW5. For each sample, 1.0 L of water was taken from the fixed depth of the water layer using a sterile bottle, and the sterile bottle containing the water sample was immediately placed on the ice [52 ], and then filtered through 0.22 μM the porous polycarbonate membrane (Millipore, MA, USA) by Shimadzu vacuum membrane pump (VP–10L). The samples were stored in liquid nitrogen until DNA was extracted. Temperature, pH, DO, Chlorophyll-a and total dissolved solids were measured by Macro-900 (Palintest, Germany) handheld multi-parameter field instrument (Additional file 1: Table S5). A total of 192 water samples were collected, and about 1000 ml of each water sample was used to measure the physicochemical factors by Macro-900. All fish were sampled using multimesh gillnets that were 18 m long, 1.5 m height with mesh sizes between 6.25 and 60 mm of the following order: 45, 20, 6.25, 10, 55, 40, 12.5, 25, 15, 60, 35, and 30 mm [53 ]. All fish were then anesthetized using the 40 mg/L of tricaine methanesulfonate (MS-222) for subsequent sampling.

Performed in deionized water using a Fastscan Dimension Icon (Bruker Corporation, Santa Barbara, CA). Images were recorded in peak force tapping mode using oxide sharpened micro-fabricated Si₃N₄ coated with Au cantilevers with a nominal spring constant of ~0.24 N/m (Bruker Corporation). Conidia were harvested from 12–15 days old malt agar slants maintained at ambient temperature, washed extensively with Tween-water, followed by washing twice with MilliQ water, subjected to PFA-fixation (Aimanianda et al., 2009 (link)) and then immobilized by mechanical trapping into porous polycarbonate membranes (Millipore). After filtering a concentrated conidial suspension, the filter was gently rinsed with deionized water, carefully cut, attached to a steel sample puck using a small piece of double face adhesive tape, and the mounted sample was transferred into the AFM liquid cell while avoiding dewetting. Data processing was performed using the commercial Nanoscope Analysis software (Bruker Corporation). At least eight conidia were analyzed to determine the percentage of conidia completely and partially covered by the surface rodlet-layer.