Introduction

Earthworms belong to the phylum of Annelida and near 700 genera of them have been recognized up to now, but estimated number of earthworm species may reach up to 7000 species (Pechenik, 2009). These worms have a cylindrical body comprised of many segments (80-190). There are specific loops at the anterior part of their body called clitellum which has a particular role in sexual reproduction of some annelids. Earthworms are regarded as one of the most important types of living creatures in the soil and play a key role in vermicompost production (Karmegam et al., 2019; Sharma and Garg, 2019). This process consists of oxidating and stabilizing organic wastes through routine activities of earthworms and other microorganisms.

Eisenia fetida (Phylum Annelida, Family Lumbricidae) is regarded as one of the most important species of earthworms and used in vermicompost process, fish feed (Musyoka et al., 2019) and poultry (Gunya et al., 2019) as well as in medicine. The protein complex of G-90 is a mixture of macromolecules with glycolipoprotein extracted from tissues of the mature worm (Grdisa and Herzenjak, 2007).

G-90 can be used for the treatment of dermal wounds due to its component involving in accelerating the cellular proliferation (Deng et al., 2018; Song et al., 2015) and enhancing the synthesis of different growth factors (e.g., fibroblast growth factor [FGF] and epidermal growth factor [EGF] in cell cultures). In fact, EGF plays its roles in this process by stimulating epithelial cells proliferation, and FGF through veins elimination, and increasing fibroblasts growth (Grdisa et al., 2004). G-90 has many bioeffects such as insulin-like protein activities accompanied with mitogenic effects (DiCicco-Bloom and Black, 1988), anti-oxidative activities, hemostasis regulation, hemolytic effects (Wang et al., 2019), bacteriostatic and bacteriolytic activities (Tutar and Karaman, 2017), anti-inflammatory and antipyretic effects (Balamurugana et al., 2009), fibrinolytic and anti-coagulant activities (Akazawa et al., 2018; Popovic et al., 2001), therapeutic effects on tumor cells (Chen et al., 2007; Cooper et al., 2004), proliferative effect on cells (Permana et al ., 2018; Herzenjak et al., 1993), and enhancing cell adhesion (Popovic et al., 1998) as well as stimulating the regeneration of peripheral nerves through Schwann cells migration (Chang et al., 2009; Bhambri et al., 2018; Moon and Kim, 2018). Lumbrokinases are a group of enzymes with molecular weights of 25-32 kDa isolated from coelomic fluid and intestinal tissue of E. fetida (Mihara et al., 1991), which has been used as a thrombolytic drug in patients with acute and chronic thrombotic disorders.

 The current study aimed to identify adult worms using molecular techniques according to the second internal transcribed spacer of the nuclear ribosomal DNA (rDNA-ITS2) and cytochrome C oxidase subunit 1 of the mitochondrial DNA (mtDNA-COX1) from adult E. fetida and to determine electrophoretic pattern of glycolipoprotein extract (G-90) for assessment of their potential biological activities in near future.

Materials and Methods

1. Preparation of G-90

The fresh E. fetida earthworms were collected from an earthworm farm in Karaj, Iran. They were kept under optimal conditions in the laboratory to breed (Lowe et al., 2014). Ten E. fetida earthworms were washed with 0.6% sodium chloride solution, to cleanse their intestine and body surface. They were cut into pieces of about 1-2 cm long and then homogenized with the homogenizer. Samples were transferred to a beaker containing methanol-chloroform solution in a 1:1 ratio and left at 4℃ overnight. Then distilled water was added until it reached the total volume of 20 ml. The mixture was centrifuged at 4000 rpm for at least 15 min. Three clearly visible layers were obtained. The upper layer was removed and methanol evaporated almost completely. The remaining sample containing G-90 was stored at -20℃ until use.

2. Electrophoretic Pattern of G-90

The extracted G-90 was loaded onto SDS-PAGE gels and ran according to Laemmli (1970). Electrophoretic patterns of the samples with different concentrations were screened under reducing conditions by discontinuous buffer system in a Mini-protean III cell apparatus (Bio-Rad) at 110v constant voltage for 60 min. SDS-PAGE was performed with a 12% resolving gel and a 5% stacking gel. A protein marker covering a wide range of molecular weights from 10-200 kDa (Fermentas, SM 0661) was used to determine the molecular weights of the proteins. The gels were stained with coomassie blue and then photographed.

3. DNA Extraction and PCR

DNA was extracted from clitellum of a single mature E. fetida using a DNA extraction kit (MBST, Iran) according to the instructions of the manufacturer. DNA was extracted using two different methods: isoamyl alcohol/chloroform and lysis techniques. The first method: isoamyl alcohol/chloroform (1:24) was added and mixed well by inversion. The contents were centrifuged at 8000 rpm for 10 min and the supernatant aqueous layer containing DNA was transferred to another centrifuge tube and 2/3rd volumes of cold isopropanol was added, mixed by inversion to precipitate the nucleic acids. All DNA samples were stored at -20℃ until further studies. DNA fragments of r-ITS2 and mt-COX1 were amplified based on the specific primer design including: EfI: (forward; 5׳-CGATGAAGAGCGCAGCCAGC-׳3) and (reverse; ׳5- CTGAGGGAATCCTTGTTAG-׳3) for ITS2, and EfC: (forward; ׳5- GAGCTAAGACAACCAGGTGC-׳3) and (reverse; ׳5- GGCTAGGTCTACTGAGGGC-׳3) for COX1. The PCR reaction was performed in a final volume of 50µl, containing 25µl of Taq master mix (Sinaclon, Iran), 2µl of each primer (10µM each), 10µl of template DNA using an automated thermocycler under the following thermal conditions: 5 min incubation at 94℃ as initial denaturation step to denature the double stranded DNA, followed by 35 cycles of 94℃ for 45s (denaturation step), 57℃ for 45s for COX1 primers and 53℃ for 45s for ITS2 primers (annealing step), and 72℃ for 45s (extension step). Lastly, the PCR was completed with a final additional extension step at 72℃ for 10 min. Samples without genomic DNA were used as negative controls and a DNA marker was used to show the length of the ITS2 and COX1 amplicons. The PCR products were electrophoresed using 1% agarose gels in 0.5x TBE buffer and thereafter visualized using Syber Safe stain (Sinaclon, Iran) on a UV illuminator. Consequently, the PCR products were purified using a quick PCR product purification kit (MBST, Iran) based on the instructions of the manufacturer.

4. Sequencing and Phylogeny

According to Sanjer׳s method, genomic DNA sequencing was performed in both directions for positive PCR amplicons by the Kawsar Biotech CO. (Tehran, Iran). The samples were assessed using Basic Local Alignment Search Tool (BLAST) and Bioedit software. The sequences were aligned and compared with each other and those of Eisenia spp. isolates as previously submitted to GenBank. The phylogenetic relationships of all these Eisenia isolates were generated by Mega 6 software based on ITS2 and COX1 sequences.

Results

The results of this study are presented in two parts comprised of protein electrophoretic pattern of G-90 complex and molecular characteristics of E. fetida.

1. Electrophoretic Pattern of G-90

The electrophoresis of the glycolipoprotein G- The electrophoresis of the glycolipoprotein G-90 showed 10 bands with approximate molecular weights of 14-130 kDa extracted from adult E. fetida (Figure 1).

Figure 1. SDS-PAGE analysis of G-90 complexextracted from the Iranian isolate of E. fetida

Lane 1: Sample (Dialysis bag), Lane 2: Sample (Filter paper), M: Marker (Fermentas-SM 0661).

2. Molecular Characteristics

2.1. DNA Extraction

Genomic DNA was extracted from a single Iranian isolate of E. fetida. The clitellum was the best part for high quality DNA extraction.The samples were loaded onto 1% agarose gel and were run for 45 minutes at a constant voltage of 100 v. Subsequently, the gel was stained with Syber Safe to check the presence of DNA in the sample. Direct lysis was a better DNA extraction method than isoamyl alcohol/chloroform one (Fig. 2A).

Figure 2. DNA was extracted from adult E. fetida and analyzed on 1% agarose gel (A). The extracted DNA was amplified using specific primers derived from COX1 (B) and ITS-2 (C) by PCR. Lane 1-2: Samples, lane C: Negative control, lane M: DNA size marker (100 bp DNA ladder).

2.2 PCR Analysis and Sequencing

PCR was performed to amplify 516 bp long ITS2 and 277 bp long COX1 fragments of E. fetida. The results are shown in Figure 2 B and C. Sequences obtained in the present study were recorded in GenBank with accession numbers of MN989855 and MN989928 for ITS2 and COXI, respectively. These sequences were compared with available data in GenBank for ITS2 (Accession numbers: EF534709.1, JX531618.1 and KU708469.1) and COX1 (Accession numbers: MH475674.1, MH475673.1, MH475672.1, MH475670.1, and MH475666.1). The BLAST analysis demonstrated that E. fetida isolate (EF534709.1) had higher similarity with our ITS2 sequence (99%), followed by JX531618.1 (94%), and KU708469.1 (88%).

COX1 nucleotide sequence showed a high similarity of 99%, compared with five isolates from GenBank (Figures 3 and 4).

All available ITS2 and COX1 nucleotide sequences were used to generate the following phylogenetic trees (Figure 5).

Figure 3. Alignment of the Nucleotide sequence of rDNA-ITS2 of the Iranian isolate of E. fetida with other sequences recorded in GenBank using reference sequences from GenBank.

Figure 4. Alignment of the Nucleotide sequence of mtDNA-COX1 of the Iranian isolate of E. fetida with other sequences recorded in GenBank using reference sequences from GenBank.

Figure 5. Phylogenetic relationships of the Iranian isolate of E. fetida based on ITS2 sequences (A) and COX1 (B) in comparison with other isolates.