Thesis (MSc)--University of Stellenbosch, 2006. / 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. / AFRIKAANSE OPSOMMING: Die moontlike ontwikkeling van geneties gebaseerde weerstand teen kadmium (Cd) by
erdwurms van die genus Eisenia, na langtermyn blootstelling aan die metaal by 'n subletale
konsentrasie, is ondersoek. Volwasse (klitellate) erdwurms van 'n langtermyn laboratorium Cdblootgestelde
bevolking (< 78 generasies) asook van ander bevolkings met geen
voorgeskiedenis van metaal blootstelling nie, is blootgestel aan 'n reeks van Cd konsentrasies
(0; 2.5; 5; 10; en 20 mg/l) in die vorm van CdSO4. Verskillende biomerkers en molekulêre
merkers is gebruik om vas te stel of die erdwurms geakklimeer of aangepas het by die metaal
in die gekontamineerde omgewing.
Akklimasie is op verskillende fisiologiese en biochemiese vlakke getoets deur die volgende
drie biomerkers te gebruik: Die 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromied
toets (MTT toets), wat mitochondriale aktiwiteit en sel lewensvatbaarheid meet; die enkel sel
elektroforese toets (Komeettoets) wat DNS string breuke meet; die biosintese van
metallotioniene (MT's) wat metaalvergiftiging verlaag. Erdwurms van die langtermyn
blootstellingsubstraat asook eksemplare van bevolkings wat nie aan kadmium blootgestel was
nie, is gebruik.
Aanpassing is ondersoek deur gebruik te maak van allosiem polimorfisme, waar 7 ensiem
koderende lokusse gebruik is asook van DNS polimorfisme deur neutrale genetiese merkers te
selekteer. Die merkers wat gebruik is, was sitochroom c oksidase, subeenheid I (COI) en
metallotionien-2 (mt-2) respektiwelik. Hierdie toetse is met dieselfde groepe bevolkings
uitgevoer. 'n Bykomende van die studie aspek waar ook van DNS polimorfisme gebruik
gemaak is, was daarop gemik om die spesie wat vir die huidige studie gebruik is, geneties te
identifiseer en om vas te stel of die erdwurms aan die spesie Eisenia fetida of Eisenia andrei
behoort. Die MT biosintese het nie die verwagte data gegenereer nie, hoofsaaklik weens die feit dat
geskikte teenligaampies nie beskikbaar was nie (bespreek in Hoofstuk 5 afdeling 5.3.3).
Soortgelyk, het die waardebepaling van DNS polimorfisme by die geselekteerde genetiese
merker mt-2 nie genoegsame informasie verskaf om die potensiaal van die ontwikkeling van
weerstand teen Cd in die langtermyn blootgestelde groep te verstaan nie. COI geenvolgordes
van E. fetida en E. andrei wat op Genbank beskikbaar was, is gebruik om met die resultate van
die huidige studie te vergelyk. Die spesie wat tydens die huidige studie gebruik is, is op hierdie
wyse geïdentifiseer as E. andrei.
Allosiem polimorfisme het geen vaste genetiese verskille tussen die langtermyn blootgestelde
laboratoriumkulture en die ander bevolkings getoon nie. Al die bevolkings het verskil van die
Hardy-Weinberg ewewigstoestand (χ2 > 5.9; p < 0.05) en het 'n lae heterosigositeit per lokus
getoon (Ho ≤ 0.21), moontlik as gevolg van inteling.
Sellewensvatbaarheid en proliferasie soos getoets met die MTT toets, het getoon dat
selomosiete, geïsoleer vanuit die langtermyn Cd blootgestelde groep, die hoogste
lewensvatbaarheid (98.42%) gehad het in vergelyking met die ander groepe (+/- 80%).
Kruksal-Wallis ANOVA (H2, 225=109.7165 p < 0.001) het getoon dat die langtermyn Cd
blootgestelde laboratoriumkultuur 'n beter respons vir akute blootstelling aan Cd gehad het.
Hierdeur is gedemonstreer dat hierdie wurms 'n soort toleransie teenoor kadmium ontwikkel
het.
Soortgelyk het die komeettoets aangetoon dat daar in die langtermyn blootgestelde eksemplare
minder DNS breuke voorgekom het na verdere blootstelling aan Cd as in die ander groepe.
Van al die komeet parameters wat tydens die studie gemeet is (komeet stertlengte, Olive stert
moment en stert DNS persentasie), het die stert DNS persentasie geblyk om die mees
sensitiefste te wees, alhoewel al drie parameters aangetoon het dat die langtermyn Cd
blootgestelde eksemplare meer weerstandbiedend was teen induksie van DNS enkelstring
breuke weens Cd blootstelling (p < 0.001), as die wat nie voorheen blootgestel was nie. Die komeet en MTT toetse het aangetoon dat erdwurms met 'n voorgeskiedenis van Cd
blootstelling 'n toenemende fiksheid teen hoër dososse van Cd ontwikkel het as die wat nie
voorheen aan kadmium blootgestel was nie. My bevindings kon hoofsaaklik toon dat verskeie
meganismes op die fisiologiese en biochemiese vlak 'n rol sou kon speel om Cd blootgestelde
bevolkings by 'n chemies stresvolle omgewing te laat akklimeer. Duidelike ondersteuning vir
die verskille tussen die getoetsde bevolkings deur van Cd geselekteerde merkers soos die mt-2
geen gebruik te maak om 'n meer finale afleiding te kan maak is nie verkry nie en behoort
verder ondersoek te word.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/22010 |
| Date | 12 1900 |
| Creators | Voua otomo, Patricks |
| Contributors | Reinecke, S.A., Swart, A., Jansen Van Vuuren, B., Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xv, 99 leaves : ill. |
| Rights | Stellenbosch University |
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