Earthworms and substrate
Two earthworm species including A. trapezoides (Weight: 930±117 mg; length: 11±1 cm: segments: 139±9) and E. fetida (Weight: 585± 70 mg; length: 9±1 cm: segments: 104±12) were collected by hand sorting from a rural area (Kaleno; N 36○ 30'; E 51○ 14') in the north of Iran and identified according to common keys, which reported by Sinkakarimi et al. (2020). These species were cultured in the laboratory by growing in sterilized host soil and cattle manure, respectively (Lowe and Butt 2007).
APPJ device
The jet plasma is produced by an APPJ device, the detailed information of this system has been described in the previous study (Hosseinzadeh Colagar et al. 2020). The specifications of this device can be summarized as follows: 99 % argon and 1 % air; gas flow, 1 l/min; a sinusoidal alternating voltage with a peak voltage of 8 kV and a frequency of 18.56 kHz; and Temperature and humidity of the room were 25˚C and 70%, respectively. The APPJ emission spectra in the wavelength range of 300-900 nm were captured and some highlighted spectral lines were shown in figure 1.
Regeneration test
Gut cleaned earthworms were rinsed with deionized water, cooled down with a wet block of ice, and amputated quickly with a sharp scalpel from 24 segments after clitellum under a dissecting microscope. Posterior segments were removed and anterior segments saved. After amputation, earthworms were transferred to sterilized media for one day to heal the wounds. Then, earthworms were anesthetized using 10% ethanol and exposed to APPJ for 0 (as control), 5, 10, 20, and 30 seconds. Host soil and cow dung respectively used for A. trapezoides and E. fetida as the substrate for culturing in polyethylene containers (2 litter). Cow dung was used as food during the test for A. trapezoides. After three months, each earthworm was placed on a block of wet ice, and the length of regenerating segments measured (mm/individual), under a dissecting microscope. These manipulations were designed to observe the effects of different treatments of APPJ on lengths of regeneration.
Molecular and biochemical response
Three gut-cleaned earthworms per treatment were homogenized together in an extraction buffer (50 mM Tris-HCl, pH 7.5; 0.25 M, Sucrose; 1mM EDTANa2, pH 7.5) in a 1/4 w/v ratio using a homogenizer (IKA T18 Ultra-Turrax, USA) for 30 s at 10000 rev/min. Homogenates were centrifuged at 10,000 rpm for 15 min in 4 ○C. After centrifugation, the supernatants were collected and stored at -80 °C until required for Molecular and biochemical analysis (Chen et al. 2011; Liu et al. 2015). Malondialdehyde content was used as a biomarker of lipid peroxidation based on the thiobarbituric acid assay according to the methods described by Ohkawa et al. (1979) with some modification. TAC was evaluated using the ferric reducing ability of plasma (FRAP) according to the method described by Benzie and Strain (1996). The content of MDA and TAC was calculated using a molar extinction coefficient of 1.56 × 105 M-1 cm-1 by measuring the absorption at 532 and 593 nm, respectively, using a spectrophotometer. Catalase activity was evaluated using H2O2 as substrate according to the Aebi method (Aebi 1984). The content of catalase was calculated using the molar extinction coefficient of 0.0394 mM-1 cm-1. Changes in absorbance were measured at 240 nm and the linear decrease in absorbance was recorded over 3 min by a spectrophotometer.
SDS-PAGE of total protein
Discontinuous sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (1979) with 15% acrylamide gels and 50 μg of the coelomic fluid proteins per lane was loaded. Staining with Coomassie Brilliant Blue R250 was used to localize the protein bands. Molecular weights were calculated according to prestained molecular weight standards. The electrophoresis run was carried out with 70 V for 3.5 h per plate towards the cathode. Bradford (1976) method was used for the determination of total protein, using by measuring absorbance at 593 nm and bovine serum albumin/BSA as a standard.
TUNEL assay
Five primary segments were separated from the irradiated plasma area and homogenated. TUNEL (terminal dUTP nick-end labelling) assay was used for the evaluation of DNA fragmentation. This test was performed by an in situ cell death detection kit (Avicenna Research Institute, Iran) according to the manufacturer's standard protocol with 200 cells for each sample.
Statistical analysis
Statistical analyses carried out using SPSS ver. 19.0 software. The normality of data was tested using the Shaprio-Wilk test. Datasets were analyzed using a general linear model (GLM) with Turkey’s pairwise comparison to assess the significant difference between groups. The statistical significance levels were set at p< 0.05.