Investigation of Some Biological Properties of Coelomic Fluid Originating from the Red Worm Eisenia fetida

Abstract

Eisenia fetida is abundant and vital member of the soil environment. The coelomic fluid they secrete help their physiological activities. This liquid, which they release during stress, also has a significant effect on soil health and ecotoxicity. In this study, some biological properties of coelomic fluid were investigated. For this purpose, 3 bacteria were selected from coelomic fluid, they were identified based on the 16S rRNA sequence and 2 of these were similar to Bacillus cereus and 1 to Bacillus thuringiensis. When the enzymatic activities in the coelomic fluid were controlled, the presence of lipase, cellulase, amylase, protease, phytase and arylsulfatase activities were detected. The antibacterial effects of the coelomic fluid was investigated against Enterococcus faecalis, Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhimurium, Yersinia enterocolitica and Pseudomonas aeruginosa. According to the results, it showed inhibitory effects on test bacteria. Additionally, the thin layer chromatography results showed the presence of glucose and maltose in the coelomic fluid. Protein pattern of fluid showed five clear protein bands in the range of 17–46 kDa. With this study, some biological properties of coelomic fluid have been revealed, and will shed light on future studies.

Keywords

• Coelomic fluid
• Earthworm
• Enzyme activity
• Protein profile
• Eisenia fetida
• Antimicrobial activity,
• Antimikrobiyal aktivite
• Sölomik sıvı
• Solucan
• Eisenia fetida
• Enzim aktivitesi
• Protein profili

Kırmızı Solucan Eisenia fetida Kaynaklı Sölomik Sıvının Bazı Biyolojik Özelliklerinin Araştırılması

Abstract

Eisenia fetida, toprak ortamının bol bulunan hayati üyelerindendir. Salgıladıkları sölomik sıvı fizyolojik aktivitelerine yardımcı olur. Stres sırasında serbest bıraktıkları bu sıvı, toprak sağlığı ve ekotoksisite üzerinde de önemli bir etkiye sahiptir. Bu çalışmada, sölomik sıvının bazı biyolojik özellikleri araştırılmıştır. Bu amaçla sölomik sıvıdan 3 bakteri seçilmiş, 16S rRNA dizilimine göre tanımlanmış ve bunlardan 2 tanesi Bacillus cereus ve 1 tanesi Bacillus thuringiensis ile benzerlik göstermiştir. Sölomik sıvıdaki enzimatik aktiviteler kontrol edildiğinde lipaz, selülaz, amilaz, proteaz, fitaz ve arilsülfataz aktivitelerinin varlığı tespit edilmiştir. Sölomik sıvının antibakteriyel etkileri Enterococcus faecalis, Escherichia coli, Staphylococcus epidermidis, Stapylococcus aureus, Klebsiella pneumoniae, Salmonella typhimurium, Yersinia enterocolitica ve Pseudomonas aeruginosa’ya karşı araştırılmıştır. Sonuçlara göre sölom bakterileri üzerinde inhibitör etkiler göstermiştir. Buna ek olarak, ince tabaka kromatografisi sonuçları ise sölomik sıvıda glikoz ve maltoz varlığını göstermiştir. Sıvının protein paterni, 17-46 kDa aralığında beş açık protein bandı göstermiştir. Bu çalışma ile sölomik sıvının bazı biyolojik özellikleri ortaya konulmuş olup, bundan sonraki çalışmalara ışık tutacaktır.

Keywords

• Eisenia fetida
• Antimikrobiyal aktivite
• Sölomik sıvı
• Solucan
• Eisenia fetida
• Enzim aktivitesi
• Protein profili
• Eisenia fetida, Enzim aktivitesi, Protein profili

References

1. Akazawa, S.I., Tokuyama, H., Sato, S., Watanabe, T., Shida, Y., Ogasawara, W. 2018. High-pressure tolerance of earthworm fibrinolytic and digestive enzymes. J. Biosci. Bioeng., 125(2): 155-159. https://doi.org/10.1016/j.jbiosc.2017.08.011
2. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J. 1990. Basic local alignment search tool. J. Mol. Bio. 215(3): 403-410. https://doi.org/10.1016/S0022-2836(05)80360-2
3. Aydilek, S. 2005. The effect of coelomic fluid of earthworms against various cells. MSc, İstanbul University, İstanbul, Turkey.
4. Bhadauria, T., Saxena, K.G. 2010. Role of earthworms in soil fertility maintenance through the production of biogenic structures. Appl. Environ. Soil Sci. https://doi.org/10.1155/2010/816073
5. Bilej, M., Brys, L., Beschin, A., Lucas, R., Vercauteren, E., et al. 1995. Identification of a cytolytic protein in the coelomic fluid of Eisenia foetida earthworms. Immun. Lett. 45: 123-128. https://doi.org/10.1016/0165-2478(94)00248-P
6. Blouin, M., Hodson, M.E., Delgado, E.A., Baker, G., Brussaard, L. et al. 2013. A review of earthworm impact on soil function and ecosystem services. Eur. J. Soil Sci. 64: 161-182. https://doi.org/10.1111/ejss.12025
7. Choi, Y.M., Suh, H.J., Kim, J.M. 2001. Purification and properties of extracellular phytase from Bacillus sp. KHU-10. J. Protein Chem. 20: 287-292. https://doi.org/10.1023/a:1010945416862
8. Clark, M.E. 1964. Biochemical studies on the coelomic fluid of Nephtys hombergi (Polychaeta: Nephtyidae), with observations on changes during different physiological states. Biol. Bull. 127(1): 63-84. https://doi.org/10.2307/1539345

9. Cooper, E.L., Ellen, K., Andrea, C. 2002. Digging forinnate immunity since Darwin and Metchnikoff. Bio Essay, 24: 319-333. https://doi.org/10.1002/bies.10077
10. Cooper, E.L., Roch, P. 2003. Earthworm immunity: a model of immune competence. Pedobiologia, 47: 676-688. https://doi.org/10.1078/0031-4056-00245
11. Demirkan, E., Aybey Çetinkaya, A., Abdou, M. 2021. Lipase from new isolate Bacillus cereus ATA179: optimization of production conditions, partial purification, characterization and its potential in the detergent industry. Turk. J. Biol. 45(3): 287-300. https://doi.org/10.3906/biy-2101-22
12. Demirkan, E., Sevgi, T., Akcakoca, D., Ersoy, F. 2017. Partial purification, characterization and wheat bran degradation studies of a new phytase from the Bacillus megaterium EBD 9-1 strain. Turk. J. Biochem. 4(3): 329-337. https://doi.org/10.1515/tjb-2016-0265 Demirkan, E., Sevgi, T., Baskurt, M. 2016. Optimization of Physical factors affecting the production of the α-Amylase from a newly Isolated Bacillus sp. M10 strain. Karaelmas Sci. Eng. J. 6(2): 1-8.
13. Devi, C., Khwairakpam, M. 2020. Feasibility of vermicomposting for the management of terrestrial weed Ageratum conyzoides using earthworm species Eisenia fetida. Environ. Technol. Innov. 18: 100696. https://doi.org/10.1016/j.eti.2020.100696
14. Engelmann, P., Kiss, J., Cooper, E.L., Nemeth, P. 2004. Earthworm leukocytes kill Hela, Hep-2, PC -12 and PA 317 cells invitro. J. Biochem. Biophys. Meth. 61: 215-228. https://doi.org/10.1016/j.jbbm.2004.04.004
15. Esaivani, C., Vasanthi, K., Singh, A.J. 2017. An Investigation on antimicrobial potency of coelomic fluid of earthworm Eudrilus eugeniae. British J. Med. Health Res. 4(4): 2394-2967.
16. Eue, I., Kauschke, E., Mohrig, W., Cooper, E.L. 1998. Isolation and characterization of earthworm hemolysins and agglutinins. Dev. Comp. Immunol. 22: 13-25. https://doi.org/10.1016/s0145-305x(97)00049-9
17. Garriga, M., Almaraz, M., Marchiaro, A. 2017. Determination of reducing sugars in extracts of Undaria pinnatifida (harvey) algae by UV-visible spectrophotometry (DNS method). Actas de Ingeniería 3: 173-179.
18. Gudeta, K., Kumari, S., Bhagat, A., Julka, J.M., Goyal, R. 2020. Mitogenic, Antioxidative and Antimicrobial Activities of G-90 and Coelomic Fluid From Earthworm As A Therapeutic Agent. Inter. J. Pharmaceut. Sci. Res. 11(11): 5298-5307. https://doi.org/ 10.13040/IJPSR.0975-8232.11(11).5298-07
19. Haque, S.F., Sen, S.K., Pal, S.C. 1995. Nutrient optimization for production of broad spectrum antibiotic by Streptomyces antibioticus SR15. 4. Acta Microbiol. Immun. Hungar. 42 (2): 155-162.
20. Kauschke, E., Pagliara, P., Stabili, L., Cooper, E.L. 1997. Characterization of Proteolytic Activity in Coelomic Fluid of Lumbricus terrestris L. (Annelida, Lumbricidae) Comp. Biochem. Physiol. Part B: Biochem. Mol. Biol. 116:235-242. https://doi.org/10.1016/S0305-0491(96)00248-9
21. Kauschke, E., Mohrig, W., Cooper, E.L. 2007. Coelomic fluid proteins as basic components of innate immunity in earthworms. Eur. J. Soil Biol. 43: 110-115. https://doi.org/10.1016/j.ejsobi.2007.08.043
22. Keay, L., Wildi, B.S. 1970. Proteinases of the genus Bacillus. I. Neutral proteinases. Biotech. Bioeng. 12: 179-212. https://doi.org/10.1002/bit.260120205.
23. Kizilkaya, R., Dengiz, O. 2010. Variation of land use and land cover effects on some soil physico-chemical characteristics and soil enzyme activity. Zemdirbyste. 97(2): 15-24.
24. Konosu, S., Chen, Y.N., Hashimoto, Y. 1966. Constituents of the extracts of a marine worm, Perinereis brevicirrus. Bull. Jpn. Soc. Sci. Fish. 32(10): 881-886.
25. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259): 680-685.
26. Lavelle, P., Chauvel, A., Fragoso, C. 1995. Faunal activity in acid soils. In: Date R.A., Grundon N.J., Rayment G.E., Probert M.E. (editors). Plant-Soil Interactions at Low pH: Principles and Management. Developments in Plant and Soil Sciences, Springer, Dordrecht, pp. 201-211. https://doi.org/10.1007/978-94-011-0221-6_29
27. Li, C., Chen, M., Li, X., Yang, M., Wang, Y., Yang, X. 2017. Purification and function of two analgesic and anti- inflammatory peptides from coelomic fluid of the earthworm, Eisenia foetida. Peptides. 89: 71-81. https://doi.org/10.1016/j.peptides.2017.01.016.
28. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275.
29. Miller, G. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428. https://doi.org/10.1021/ac60147a030
30. Milochau, A., Lassègues, M., Valembois, P. 1997. Purification, characterization and activities of two hemolytic and antibacterial proteins from coelomic fluid of the annelid Eisenia fetida andrei. Biochim. Biophys. Acta (BBA)-Protein Struc. Mol. Enzym. 1337(1): 123-132. https://doi.org/10.1016/S0167-4838(96)00160-4
31. Mishra, P.K., Bisht, S.C., Jeevanandan, K., Kumar, S., Bisht, J.K., Bhatt, J.C. 2014. Synergistic effect of inoculating plant growth- promoting Pseudomonas spp. and Rhizobium leguminosarum-FB1 on growth and nutrient uptake of rajmash (Phaseolus vulgaris L.). Arch. Agron. Soil Sci. 60: 799-815. https://doi.org/10.1080/03650340.2013.843773
32. Msakni, S. 2020. Screening of cellulase enzyme production by Bacillus sp. isolated from Turkish soils, nutritional optimization, partial purification of enzyme, characterization and effectiveness of plant waste removal in wool fabric. MSc, Bursa Uludağ University, Bursa, Turkey.
33. Nadana, G.R.V., Selvaraj, K., Sivakumar, P., Palanichelvam, K. 2020. Coelomic fluid of earthworms extruded by cold stress method has commercially significant compounds and trigger seed germination in Vigna radiata L. Environ. Technol. Innov. 19: 100814. https://doi.org/ 10.1016/j.eti.2020.100814
34. Ohresser, M., Matagne, R.F., Loppes, R. 1997. Expression of the arylsulfatase reporter gene under the control of the nit 1 promoter in Chlamydomonas reinhardtii, Curr. Gen. 31: 264-271. https://doi.org/10.1007/s002940050204
35. Patil, S.R., Biradar, P.M. 2017. Earthworm’s coelomic fluid: extraction and importance. Int. J. Adv. Sci. Res. 2(2): 1-4.
36. Ramian, P., Arabi, M., Hemmati, R. 2018. Novel Amylase in Coelomic Fluid and Body Extract from the Earthworm Allolobophora Chlorotica. Biomacromol. J. 4(1): 35-45.
37. Robyt, J.F., White, B.J. 1987. Biochemical techniques, Theory and practice. Wadsworth. Inc., Belmont, California, USA.
38. Rochfort, S., Wyatt, M.A., Liebeke, M., Southam, A.D., Viant, M.R. et al. 2017. Aromatic metabolites from the coelomic fluid of Eisenia earthworm Species. Eur. J. Soil Biol. 78: 17-19. https://doi.org/10.1016/j.ejsobi.2016.11.008
39. Saitou, N., Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evo. 4(4): 406-425. https://doi.org/10.1093/oxfordjournals.molbev.a040454
40. Scherbert, I., Messner, B. 1997. Untersuchugen uber das vor kommenvon lysozyme bei anneliden. Zool. Jahr. Physiol. Biol. 76: 36-50.
41. Scheu, S. 2003. Effects of earthworms on plant growth: patterns and perspectives. Pedobiologia, 47(5): 846–856.
42. Sevgi, T., Demirkan, E. 2021. Evaluation of the effects of temperature, light, and UV-C radiation on HSP70A expression in Chlamydomonas reinhardtii. Turk. J. Bot. 45: 671-680. https://doi.org/10.3906/bot-2012-43
43. Sevinc, N., Demirkan, E. 2011. Production of protease by Bacillus sp. N-40 isolated from soil and its enzymatic properties. J. Biol. Environ. Scie. 5(14): 95-103.
44. Shen, Y. 2010. Earthworms in Traditional Chinese Medicine. Zoology in the Middle East, 51 (sup2): 171-173. https://doi.org/10.1080/09397140.2010.10638470.
45. Stahl, E. (Ed.). 1965. Thin-layer chromatography: a laboratory handbook. Springer Science and Business Media.
46. Sugihara, A., Tani, T., Tominaga, Y. 1991. Purification and characterization of a novel thermostable lipase from Bacillus sp. J. Biochem.109(2): 211-216.
47. Tsukamoto, K., Ariki, S., Nakazawa, M., Sakamoto, T., Ueda, M. 2021. Novel cold-adapted raw-starch digesting α-amylases from Eisenia fetida: Gene cloning, expression, and characterization. Biotechol. Rep. 31: e00662. https://doi.org/10.1016/j.btre.2021.e00662
48. Tutar, U., Karaman, İ. 2017. Investigation of antibacterial properties of mucus and coelomic fluid obtained from Eisenia fetida. Cumhuriyet Sci. J. 38(3):427-434. https://doi.org/10.17776/csj.340474
49. Murugan, S., Umamaheswari, S. 2021. Identification of lysenin protein function in coelomic fluid of Eudrilus Eugeniae. Eur. J. Mol. Clin. Med. 8(3): 2021.
50. Valembois, P., Lassegues, M., Roch, P. 1992. Formation of brown bodies in the coelomic cavity of the earthworm Eisenia foetida Andrei and attendant changes in shape and adhesive capacity of constitutive cells. Develop. Comp. Immun. 16: 95-101. https://doi.org/10.1016/0145-305x(92)90010-a
51. Vasantha, N.C., Rajagopalan, K., Selvan Christyraj, J.D., Subbiahanadar Chelladurai, K., Ganesan, M. et al. 2019. Heat-inactivated coelomic fluid of the earthworm Perionyx excavatus is a possible alternative source for fetal bovine serum in animal cell culture. Biotechnol. Prog. 35(4): e2817. https://doi.org/10.1002/btpr.2817
52. Vasanthi, K., Chairman, K., Ranjit Singh, A.J.A. 2013. Antimicrobial activity of earthworm (Eudrilus eugeniae) paste. Afr. J. Environ. Sci. Technol. 7(8): 789-793. https://doi.org/10.5897/AJEST2013.1455
53. Wang, C., Sun, Z., Zheng, D. 2006. Research advance in antibacterial immunity ecology. J. Appl. Ecol. 17(3): 525-529.
54. Wang, C., Sun, Z.J., Liu, Y.Q., Zheng, D.M., Liu, X.L. et al. 2007. Earthworm polysaccharide and its antibacterial function on plant-pathogen microbes in vitro. Eur. J. Soil Biol. 43: 135-142. https://doi.org/10.1016/j.ejsobi.2007.08.035.
55. Yakkou, L., Houida, S., Dominguez, J., Raouane, M., Amghar, S. et al. 2021. Identification and characterization of microbial community in the coelomic fluid of earthworm (Aporrectodea molleri). Microbiol. Biotechno. Lett. 49(3): 391-402. https://doi.org/10.48022/mbl.2104.04013
56. Yamaji, A., Sekizawa, Y., Emoto, K., Sakuraba, H., Inoue, K., et al. 1998. Lysenin, a novel sphingomyelin-specific binding protein. J. Biol. Chem. 273: 5300-5306. https://doi.org/10.1074/jbc.273.9.5300
57. Yoo, Y.J., Hong, J., Hatch, R.T. 1987. Comparison of a-amylase activities from different assay methods. Biotechnol. Bioeng. 147-51. https://doi.org/10.1002/bit.260300120
58. Yousra, B. 2018. Diversity in the susceptibility of Botrytis cinerea strains to the biological control agent Pseudomonas helmanticensis. IOBC/WPRS Bull. 133:100-104.
59. Yuk, J., Simpson, M.J., Simpson, A.J. 2012. Coelomic fluid: a complimentary biological medium to assess sub-lethal endosulfan exposure using 1 H NMR-based earthworm metabolomics. Ecotoxicol. 21(5): 1301-1313. https://doi.org/10.1007/s10646-012-0884-5