Molecular characterization and DNA methylation profile of Libyodrilus violaceous from oil polluted soil

Document Type: Original article


1 Department of Biological Sciences, Redeemer’s University, Ede Osun state, Nigeria

2 School of Life Sciences, Discipline of Microbiology, University of Kwazulu-Natal, Durban, South Africa

3 Department of Biotechnology Engineering, Ambala college of Engineering and Applied Research, Devsthali, P.O Sambhalkha, Ambala-133101, Haryana, India

4 School of Life Sciences, Discipline of Genetics, University of Kwazulu-Natal, Durban, South Africa


Studies on earthworms using molecular markers are rare in Africa except a handful from South Africa. Reports on Libyodrilus violaceous,an earthworm found in West Africaare available including their metal tolerance and bioaccumulation capacity but their molecular characterization and ecotoxicology studies are scarce. In this study, triplicate L. violaceous specimens were collected from four locations within a petroleum polluted site and one in a control site, ≃1Km away from point of spill. DNA was extracted and 18S rRNA and 16S rRNA genes were amplified and sequenced. DNA methylation of their 18S rRNA gene was determined using Methylation specific PCR (MSP) method.  Phylogenetic trees generated for 18S rRNA and 16S rRNA genes grouped L. violaceous within the Eudrilidae family concurrent with its conventional grouping and MSP results indicate no methylation in L. violaceous population from this site.


1. Huang J, Xu Q, Sun ZJ, Tang GL, Su ZY. Identifying earthworms through DNA barcodes. Pedobi 2007;51:301-309.
2. Hendrix PF, Callaham Jr MA, Drake JM, Huang CY, James SW, Snyder BA, Zhang W. Pandora's box contained bait: the global problem of introduced earthworms. Annu Rev Ecol  Syst 2008;39:593-613.
3. Szederjesi T, Vavoulidou E, Chalkia C, Dányi L, Csuzdi C. An annotated checklist of earthworms of Greece (Clitellata: Megadrili). Zootaxa 2017;4272:57-82.
4. Trakić T, Valchovski H, Stojanović M. Endemic earthworms (Oligochaeta: Lumbricidae) of the Balkan Peninsula: a review. Zootaxa 2016;4189:251-274.
5. James SW, Gamiette F. New species of DichogasterBeddard, 1888 (Clitellata: Benhamiidae) with additional records of earthworms from Guadeloupe (French West Indies). Zootaxa 2016;4178;391-408.
6. Nguyen TT, Nguyen AD, Tran BT, Blakemore RJ. A comprehensive checklist of earthworm species and subspecies from Vietnam (Annelida: Clitellata: Oligochaeta: Almidae, Eudrilidae, Glossoscolecidae, Lumbricidae, Megascolecidae, Moniligastridae, Ocnerodrilidae, Octochaetidae). Zootaxa 2016;4140:1-92.
7. Palumbi AR, Cipriano F. Species identification using genetic tools: the value of nuclear and mitochondrial gene sequences in whale conservation. J Hered 1998;89:459-464.
8. Symondson WO. Molecular identification of prey in predator diets. Mol Ecol 2002;11:627-641.
9. Yadav S, Mullah M. A review on molecular markers as tools to study earthworm diversity. Int J Pure Appl Zool 2017;5:62-69.
10. Sperling F. DNA barcoding: deuset machine. Newsl Biol Surv Canada (Terr. Arthropods). 2003;22:50-53.
11. Will KW, Rubinoff  D. Myth of the molecule: DNA barcodes for species cannot replace morphology for identification and classification. Cladistics 2004;20:47-55.
12. Szederjesi T, Pop VV, PavlÍČek T, MÁrton O, KrÍzsik V, Csuzdi C. Integrated taxonomy reveals multiple species in the Dendrobae nabyblica (Rosa, 1893) complex (Oligochaeta: Lumbricidae). Zool J Linn Soc 2017;182:500-516.
13. Hebert PD, Cywinska A, Ball SL, De Waard JR. Biological identifications through DNA barcodes. Proc Biol Sci 2003;270:313-321.
14. Wu S, Xiong J, Yu Y. Taxonomic resolutions based on 18S rRNA genes: a case study of subclass copepoda. PLoS One 2015;10:e0131498.
15. Stackebrandt E, Goebel BM. Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 1994;44:846-849.
16. Pop AA, Cech G, Wink M, Csuzdi C, Pop VV. Application of 16S, 18S rDNA and COI sequences in the molecular systematics of the earthworm family Lumbricidae (Annelida, Oligochaeta). Eur J Soil Biol 2007;43:S43-S52.
17. Klarica J, Kloss-Brandstätter A, Traugott M, Juen A. Comparing four mitochondrial genes in earthworms-implications for identification, phylogenetics, and discovery of cryptic species. Soil Biol Biochem 2012;45:23-30.
18. Bird A. DNA methylation patterns and epigenetic memory. Genes Dev 2002;16:6-21.
19. Wrobel K, Wrobel K, Caruso JA. Epigenetics: an important challenge for ICP-MS in metallomics studies. Anal Bioanal Chem 2009;393:481-486.
20. Barter MJ, Bui C, Young DA. Epigenetic mechanisms in cartilage and osteoarthritis: DNA methylation, histone modifications and microRNAs. Osteoarthritis Cartilage 2012;20:339-349.
21. Kille P, Andre J, Anderson C, Angx HN, Bruford MW, Bundy JG, Donnelly R, Hodson ME, Juma G, Lahive E, Morgan AJ, Sturzenbaum SR, Spurgeon DJ. DNA sequence variation and methylation in an arsenic tolerant earthworm population. Soil Biology and Biochemistry2013;57:524-532.
22. Gross MC, Schneider CH, Valente GT, Martins C, Feldberg E. Variability of 18S rDNA locus among Symphysodon fishes: chromosomal rearrangements. J Fish Biol 2010;76:1117-1127.
23. Li Z, Chen X, Li S, Wang Z. Effect of nickel chloride on Arabidopsis genomic DNA and methylation of 18S rDNA. Electronic Journal of  Biotechnology 2015;18:51-57.
24. Claus C. 1880. Main features of zoology. Fourth completely reworked and improved edition. Marburg: N.G. Elwert'scheVerlagsbuchhandlung.
25. Plisko JD, Nxele TC. An annotated key separating foreign earthworm species from the indigenous South African taxa (Oligochaeta: Acanthodrilidae, Eudrilidae, Glossoscolecidae, Lumbricidae, Megascolecidae, Microchaetidae, Ocnerodrilidae and Tritogeniidae). African Invertebrates 2015;56:663-708.
26. Beddard FE. Memoirs: On the structure of an earthworm allied to Nemertodrilus, Mich., with observations on the post-embryonic development of certain organs. J Cell Sci 1891;2:539-585.  
27. Sims RW, Gerard BM. Earthworms: keys and notes for the identification and study of the species Brill Archive. 1985;Vol 31.
28. Ogunlaja A, Morenikeji OA. Species diversity and population size of earthworms after oil spillage in a pipeline vandalized area in Lagos State, Nigeria. Afr J Agri Res 2013;8: 10-19.
29. Owa SO. A new earthworm genus (Eudrilidae: Oligochaeta) from Nigeria. J Nat Hist 1995; 29:571-579.
30. Bamgbose O, Odukoya OO, Arowolo TOA. Earthworms bioindicators of metal pollutions in dumpsites of Abekuta city, Nigeria. Rev Biol Trop 2000;48:1-13. 
31. Ogunlaja A. Earthworm ecology, heavy metals and total petroleum hydrocarbon assessment of an oil spill site in Agaye, Lagos State, Nigeria. Doctoral dissertation, University of Ibadan, Nigeria 2014. 
32. Dada EO, Njoku KL, Osuntoki AA, Akinola MO. Evaluation of the responses of a wetland, tropical earthworm to heavy metal contaminated soil. Analysis 2013;1:47-52.
33. Idowu AB, Edema MO, Adeyi AO. Distribution of bacteria and fungi in the earthworm Libyodrillus violaceous (Annelida: Oligochaeta), a native earthworm from Nigeria. Rev Biol Trop 2006;54:49-58.
34. Dedeke GA, Owa SO, Olurin KB, Akinfe AO, Awotedu OO. Partial replacement of fish meal by earthworm meal (Libyodrilus violaceus) in diets for African catfish, Clariasgariepinus. Int J Fish Aquac 2013;5:229-233.
35. Ogunlaja A, Ogunlaja OO. Stress indicators and behavioural end-points of Libyiodrillus violaceous exposed to petroleum products contaminated soil. J Environ Anal Toxicol 2012; 2:1-4.
36. Bamidele JA, Idowu AB, Ademolu KO, Atayese AO. Microbial diversity and digestive enzyme activities in the gut of earthworms found in sawmill industries in Abeokuta, Nigeria. Rev Biol Trop 2014;62:1241-1249.
37. Ossai EK, Iwegbue CM, Nwajei GE. Trace Elements in Water, Soil, Earthworm and Fishes from Otokutu End of Warri River, Delta State, Nigeria. Pak J Biol Sci 2014;17:1136-1140.
38. Dedeke GA, Owagboriaye FO, Adebambo AO, Ademolu KO. Earthworm metallothionein production as biomarker of heavy metal pollution in abattoir soil. Appl Soil Ecol 2016;104: 42-47.
39. Ojo OF, Adewumi DG, Oluwatoyin AK. Glutathione-S-transferase production in earthworm (Annelida: Eudrilidae) as a tool for heavy metal pollution assessment in abattoir soil. Rev Biol Trop 2016;64:779-789.
40. Dedeke GA, Iwuchukwu PO, Aladesida AA, Afolabi TA, Ayanda IO. Impact of heavy metal bioaccumulation on antioxidant activities and DNA profile in two earthworm species and freshwater prawn from Ogun River. Sci Total Environ 2018;624:576-585.
41. Bienert F, De Danieli S, Miquel C, Coissac E, Poillot C, Brun JJ, Taberlet P. Tracking earthworm communities from soil DNA. Mol Ecol 2012;21:2017-2030.
42. Hadziavdic K, Lekang K, Lanzen A, Jonassen I, Thompson EM, Troedsson C. Characterization of the 18S rRNA gene for designing universal eukaryote specific primers. PLoS One 2014;9:e87624.
43. Katoh K, Misawa K, Kuma KI, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic acids Res 2002;30:3059-3066.
44. Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 2015; 32:268-274.
45. Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS. Model Finder: fast model selection for accurate phylogenetic estimates. Nat Methods 2017;14:587-589.
46. Minh BQ, Nguyen MAT, Haeseler AV. Ultrafast approximation for phylogenetic bootstrap. Mol Biol Evol 2013;30:1188-1195.
47. James SW, Davidson SK. Molecular phylogeny of earthworms (Annelida: Crassiclitellata) based on 28S, 18S and 16S gene sequences. Invertebrate Systematics 2012;26:213-229.
48. Blakemore RJ.The major megadrile families of the world reviewed again on their taxonomic types (Annelida: Oligochaeta: Megadrilacea). Opusc Zool Budapest2013;44:107-127.
49. Kinberg JGH, Annulata nova, Ofersigt af Kungl. Ventenskaps-Akademiens Forhandlingar 1866, Stockholm 1867;23:97-103.
50. Michaelsen W. Oligochaeta. Das Tierreich 1900;10:1-575.
51. Plisko JD. An annotated checklist of the South African Acanthodrilidae (Oligochaeta: Acanthodrilidae: Acanthodrilinae, Benhamiinae). Zootaxa 2012;3458:4-58.
52. Plisko JD. A new family Tritogeniidae for the genera Tritogenia and Michalakus, earlier accredited to the composite Microchaetidae (Annelida: Oligochaeta). Afr Invertebr 2013;54; 69-93.
53. Plisko JD. Michalakus, a remarkable new genus of microchaetid earthworm from South Africa (Oligochaeta: Microchaetidae). Ann Natal Mus 37;287-293.
54. Cognetti de Martiis L. The oligochaetes of the neotropicale region. Pt.1. Mem Reale Ace Sci Torino 1905;55:1-72.
55. Benham WB.  A new English genus of aquatic Oligochaeta  (Sparganophilus) belonging  to the family Rhinodrilidae. Quart J Micros Sci 1892;34:155-179.   
56. Jamieson BGM, Tillier S, Tillier A, Justine JL, Ling E, James SW, MacDonald KR, Hugall AF. Phylogeny of the megascolecidae and crassiclitellata (Annelida, Oligochaeta): combined versus partitioned analysis using nuclear (28S) and mitochondrial (12S, 16S) rDNA. Zoosystema 2002;24:707-734. 
57. Omodeo P. Evolution and biogeography of megadriles(Annelida, Clitellata). Ital J Zool 2000;67:179-201.
58. Chang CH, Shen HP, Chen JH. Earthworm fauna of Taiwan. National Taiwan University Press. 2009 
59. Noel S, Rath SK. Randomly amplified polymorphic DNA as a tool for genotoxicity: an assessment. Toxicol Ind Health 2006;22:267-275.
60. Ogunlaja A, Ogunlaja OO, Okewole DM and Morenikeji OA. Risk assessment and source indentification of heavy metal contamination by multivariate and hazard index analysis of a pipeline vandalized area in Lagos State, Nigeria. Sci Tot Env 2019;651:2943-2952.
61. Fukushi K, Sasaki M, Sato T, Yanase N, Amano H, Ikeda H. A natural attenuation of arsenic in drainage from an abandoned arsenic mine dump. Appl Geochem 2003;18:1267-1278.
62. Mulligan CN, Yong RN. Natural attenuation of contaminated soils. Environ Int 2004;30: 587-601.