It300

Conidia production was estimated for CHL and MET leaf, stem, and root extract treated cultures (500 µg/mL). From the eight replicates of each condition, four mycelia were collected from the CCM microplates used in the AF accumulation assay, and individually washed three times in a 0.1% (v/v) Tween20^® aqueous solution by vortexing 1 min. The spore suspensions were then washed three times with a 80% (v/v) ethanol solution and conidia concentration was then determined with a Burker chamber. The remaining four CHL and MET leaf, stem, and root extract treated cultures from AF accumulation plates were observed using a Scanning Electron Microscope (SEM) JEOL IT 300, in high vacuum mode. Samples were fixed for 4 h in 3% (v/v) glutaraldehyde, acetone dehydrated (from 30% to 100% water/acetone solutions), critical point dried, and coated with a thin layer of gold by means of a Sputter Coater “Agar”. Observations were conducted at an acceleration voltage of 10.0 kV and at a 450× magnification.

For the histochemical analyses, fresh lateral and ventral mucus from four individuals was collected on standard SEM stubs, consisting of aluminium and copper (Co. Gröpl, Austria) until the whole stub was covered. All samples were then air‐dried and analysed using energy dispersive x‐ray spectroscopy (EDX) with the x‐ray microanalysis software (Software Team, Version 4.3, Co. Ametek, Germany) in the SEM JEOL IT 300 at 20 kV. The collecting time for the elements in the mucus was set to 30 s for the selected areas and 4 hr for dot mapping, both with a ~ 30% dead time. At least 5–7 point measurements from each mucus type (lateral and ventral) and each of the four individuals were taken, and these are summarised in Table 2 as a range between minimum and maximum values. For the comparison of the different mucus types, atom % (at.%) was used, values below 0.1 at.% are ignored due to the detection limit of this semiquantitative method.

Twenty‐seven specimens from 11 different localities were collected for genome skimming including nine different Penetrantia and 18 additional ctenostome specimens (Table 1). One additional penetrantiid specimen (Penetrantia sp.) was collected in Helgoland, Germany (54°08.339′ N 7°52.298′ E) for sanger sequencing of the cytochrome c oxidase subunit I (cox1) gene (OR632352) and genetic distance analysis only (see below). Samples were either collected in the intertidal zone by hand or in shallow subtidal areas by dredging. All samples were fixed either in 96% or absolute ethanol and stored at 4°C until further investigation. Stereomicroscopic pictures were taken with a Nikon SMZ25 stereomicroscope (Nikon, Tokyo, Japan) equipped with a DsRi2 microscope camera, or with a Hirox RH‐2000 3D digital microscope (Hirox Co., Ltd., Tokyo, Japan). Scanning electron microscopic images were generated using a JEOL IT 300 (JEOL, Akishima, Tokyo, Japan) with a secondary detector at 10–25 KeV.

Scanning electron microscopic images were obtained from an FEI Quanta 200 SEM (FEI, Hillsboro, OR, USA) and Jeol IT-300 tungsten scanning electron microscopy equipped with secondary backscattering electron detectors and an Oxford energy dispersive X-ray (EDX) analysis external probe. Samples were coated with gold using a Quorum Q150R sputter coater (Quorum Technologies Ltd, East Sussex, UK). The coating thickness was precisely controlled at 5 nm using the film thickness monitor option of the Quorum Q150R sputter. Functional group analysis on the surface of adsorbents was obtained from a PerkinElmer Spectrum 400 FT-IR/FT-NIR spectrometer (Waltham, MA, USA) recording from 4000 to 500 cm⁻¹. Thermal analysis of adsorbents was elucidated using a PerkinElmer TGA 4000 thermogravimetric analyzer (Waltham, USA). Centrifugation was accomplished employing a CL10 ThermoScientific centrifuge (Johannesburg, South Africa).

After fixation, the samples were washed 5 times with PBST [1× PBS, 0.05% (vol/vol) Tween-20] for 10 min. The samples were blocked in 5% BSA in PBST for 1 h at RT. Primary antibody (1:500 dilution, mouse Anti-α-Tubulin Cat # T9026, Sigma-Aldrich) incubation was performed in a blocking solution (1% BSA in PBST) for 24–36 h at 4 °C. The samples were washed with PBST for 5 × 5 min, after which samples were incubated with secondary antibodies (1:250 dilution; Goat anti-Mouse IgG Alexa Fluor 594 Cat # A-11032, ThermoFisher) diluted in blocking solution for overnight at 4 °C. Then, the samples were washed with PBST for 5 × 10 min. Imaging was performed on Leica TCS SP8 DLS and Leica DMi8 confocal microscopes. Sample preparation for scanning electron microscopy was performed as in (Kraus et al. 2016 (link)); SEM imaging was performed using the JEOL IT 300 scanning electron microscope.

To investigate the Ni adsorption as well as fluctuations in Ni caused by the different treatments along the root tip, energy dispersive X-ray spectroscopy (EDX) was employed. Fresh root tips (1 cm) were affixed with sticky carbon foil on aluminum stubs and air dried at room temperature for 24 h. The dry samples were carbon-coated (Leica Med 020). The root tips were then analyzed in a scanning electron microscope (SEM) (Jeol IT 300) and semi-quantitative measurements of element contents were taken as line scans along the sample, starting from the tip and traveling over a distance of at least 600 μm in 5-μm steps. The magnification was set to × 160. The detector and supported TEAM software were both from the EDAX company.