Browsing by Author "Voua otomo, Patricks"
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- ItemThe effects of long-term exposure to cadmium on the earthworm Eisenia Fetida (Oligochaeta) : an investigation of the development of genetic metal resistance(Stellenbosch : Stellenbosch University, 2006-12) Voua otomo, Patricks; Reinecke, S. A.; Swart, Amanda C.; Jansen van Vuuren, Bettine; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.ENGLISH ABSTRACT: The development of potential genetically based resistance to cadmium (Cd) after long-term exposure to this metal at a sublethal concentration, was investigated in earthworm specimens belonging to the genus Eisenia. Adult (clitellate) earthworms from a long-term laboratory Cdexposed population (> 78 generations) and from other populations having no previous history of metal exposure were exposed to increasing concentrations (0; 2.5; 5; 10 and 20 mg/l) of Cd in the form of CdSO4. Different biomarkers and molecular markers were used to determine whether the specimens from the long-term Cd-exposed population had acclimatized or adapted to the metal contaminated environment. Acclimation was investigated at different physiological and biochemical levels using the following three biomarkers: the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT assay) measuring mitochondrial activity and cell viability; the single cell gel electrophoresis assay (comet assay) measuring DNA strand breaks and the biosynthesis of metallothioneins (MTs) that have the function of lowering metal toxicity. Earthworms from the long-term exposed substrate as well as specimens from populations not exposed to Cd were used. Adaptation was investigated by looking at both allozyme polymorphism at seven enzyme encoding loci and DNA polymorphism using chosen neutral and selectable genetic markers. The markers used were cytochrome c oxydase subunit I (COI) and metallothionein-2 (mt-2) respectively. This was done amongst the same pool of populations. Additionally, the DNA polymorphism study also aimed at genetically identifying the species utilized in this study thereby determining whether these earthworms belong to the species Eisenia fetida or Eisenia andrei. MTs biosynthesis did not generate the expected data mainly due to the fact that a suitable antibody could not be obtained (discussed in Chapter 5 section 5.3.3.). Similarly, the assessment of DNA polymorphism at the chosen selectable genetic marker mt-2 did provide information relevant to understanding the potential development of resistance to Cd in the long-term metal contaminated group. COI sequences generated in this study were compared to E. fetida and E. andrei COI sequences available on Genbank. Consequently, specimens used in this study were identified as possibly belonging to the species E. andrei. Allozyme polymorphism revealed no fixed genetic differences between the long-term Cd exposed laboratory culture and the rest of the populations. All the populations departed from Hardy-Weinberg equilibrium (χ2 > 5.9; p < 0.05), and showed a low mean heterozygosity per locus (Ho ≤ 0.21), probably due to inbreeding. Cell viability and proliferation as tested by the MTT assay revealed that coelomocytes, isolated from the long-term Cd-exposed group showed the highest viability (98.42%) compared to those from other groups (+/- 80%). Kruskal-Wallis ANOVA (H2, 225=109.7165 p < 0.001) revealed that the long-term Cd-exposed laboratory culture showed a better response to acute exposure to Cd, thus demonstrating that these worms have developed some kind of tolerance to Cd. Similarly, the comet assay showed that in the long-term Cd-exposed specimens, less DNA breaks occurred after Cd exposure than in the unexposed groups. Of all the comet parameters assessed in this study (comet tail length, tail moment and tail DNA percentage), tail DNA percentage seemed to be more sensitive although all three parameters indicated that long-term Cd-exposed specimens were more resistant than unexposed specimens as shown by the number of single strand DNA breaks induced by exposure to higher concentrations of Cd (p < 0.001). The comet and the MTT assays indicated that the earthworms with a previous history of Cd exposure have developed increased fitness towards higher doses of Cd, compared to previously unexposed groups. These findings mainly proved that several mechanisms could come into play at the physiological and biochemical level to allow the Cd exposed population to acclimatize to its chemically stressful environment. Clear genetic support for the differences found between the tested populations was not obtained, but needs to be investigated further using Cd selectable markers such as the mt-2 gene, in order to come to a more conclusive deduction.