Aspergillus niger

Enzymatic hydrolysis was performed by mixing 6 g (dry wt.) of pretreated ESBP with 60 mL of phosphate buffer (0.05 M, pH 5) in an Erlenmeyer flask (250 mL) and sterilized in an autoclave (120 °C for 20 min). The following enzyme activities of commercial enzymes cocktails were added to the flask: 3.2 FPU per gram of dry biomass (cellulase from Trichoderma reesei, Celluclast^®, Sigma, Darmstadt, Germany), 46.1 U of xylanase per gram of dry biomass (from Thermomyces lanuginosus, Sigma), and 46.5 U of exo-polygalacturonase per gram of dry biomass (from Aspergillus niger, Sigma). The flask was incubated at 50 °C and 150 rpm for 48 h. Samples were taken throughout the process and stored at –20 °C until use. Each experiment was carried out in triplicate.

The formation of NETs was quantified in black 96 well plates after challenging 10⁶ PMNs/ml with 10⁷ microorganisms/ml cultivated with or without 10⁵ hDFSCs/ml (MOI = 100). Control stimulation of PMNs was realized using glucose oxidase (20 mU, from Aspergillus niger, Sigma Aldrich). After 165 min of aerobic incubation extracellular DNA was quantified via adding 30 μl Sytox green (50 μM, Life Technologies, Carlsbad, California) to the samples. After 15 min of incubation (37 °C, 5% CO₂) fluorescence was measured at 485/520 nm.

Compound 4 (250 μg) was dissolved in 2.5 mL of 0.1 M sodium acetate buffer, pH 4.5 and crude pectinase from Aspergillus niger (50 μL, Sigma-Aldrich, P2736) was added. The mixture was stirred at 50 °C for 48 h. The product precipitated out during the reaction and was filtered and then crystallized. The resulting product obtained from the hydrolysis was identified as isosteviol, characterized by comparison of its co-TLC with standard compound and ¹H-NMR spectral data [19 –21 ].

To test if Phasmatodea guts were pectinolytic, we filled square Petri-dishes with 0.1% solutions of either citrus pectin (Sigma) or polygalacturonic acid (PGA) (Megazyme) in 0.4% agarose and 50 mM citrate-phosphate buffer (pH 5.0). We made wells in the plates and filled them with 5 μL of macerated, whole midguts cleared of their contents and dissected from the aforementioned six species with the published midgut transcriptomes7 (link): Aretaon asperrimus (Heteropteryginae), Peruphasma schultei (Pseudophasmatinae), Sipyloidea sipylus (Necrosciinae), and Extatosoma tiaratum (Lanceocercata: Extatosomatinae), Medauroidea extradentata, and Ramulus artemis (Clitumninae). We used pectinases from Aspergillus niger (Sigma) as positive control. Plates were incubated upside-down at 40 °C overnight, stained for one hour in 0.01% Ruthenium Red (Colour Index No. 77800) on a shaker at 20 rpm, and destained in diH2O. Enzyme activity was detectable as clearings in the stained gel.

Cytogenetic analyses were performed on samples of both induced allotetraploids, IpaDur1 S₁₀ (10th generation of self-pollination), IpaDur2 S₂ (2nd generation of self-pollination). Root tips were isolated from at least five plants of each IpaDur2 and IpaDur1. At least 10 metaphases of each plant (over 250 metaphases per genotype) were observed. Root tips (5–10 mm long) from 4-week-old plants were collected, treated with 2 mM 8-hydroxyquinoline for 2 h at room temperature and another hour at 4^°C with new solution, and then incubated in fixative solution (absolute ethanol: glacial acetic acid, 3:1, v/v) for 12 h at 4°C. Somatic chromosome spreads were prepared according to Schwarzacher and Heslop-Harrison (2000) with few modifications: meristems were digested in 10 mM citrate buffer containing 2% cellulase (from Trichoderma viridae; Onozuka R-10 Serva) and 20% pectinase (from Aspergillus niger, Sigma) for 2 h at 37°C. Chromosomes of each root were set on a slide, in a drop of acetic acid 45% and the spread was obtained by applying pressure to coverslip. Slides were selected using phase contrast in the AxiosKop microscope (Zeiss, Oberkochen, Germany). Coverslips were removed, slides were air-dried for 24 h and kept at −20°C.