The effects of long-term exposure to cadmium on the earthworm Eisenia Fetida (Oligochaeta) : an investigation of the development of genetic metal resistance

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.