Variability patterns and genetic determination of the tolerance to metal-rich acid mine drainage by planktonic invertebrates

摘要

Genetic erosion in natural populations due to pollution is an emergent problem, with repercussion in the future of these populations, since lower genetic variability difficults its ability to adapt to new environmental changes. Organism’s response to a particular stressor may occur at the behavioral level, by avoidance from contaminated sites, or at the metabolic level, by hormonal or enzymatic adjustments. More sensitive genotypes might be eliminated if the stressor works as a selective pressure. Studies with populations inhabiting at contaminated sites exhibited loss of genetic diversity. However, when genetic analysis are included in the evaluation of genetic variability, some cases revealed a lack of agreement between the two approaches i.e., evaluating a population through selectable markers, genetic erosion was detected, while, through neutral markers, did not significant decreased. To clarify this lack of agreement, it is necessary to take into account other variables. A population genetic variability is not weakened only by natural selection, but also by genetic drift, inbreeding and emigrations. Furthermore, mutations and immigrations may increase the genetic variability. This work aimed at evaluating the influence of factors that justify the occurrence of genetic erosion without significant loss of genetic variability in zooplankton populations exposed to an acidic effluent enriched in metals. Selectable markers and neutral markers were used for the evaluation of genetic variability in a copepod natural population (Copidodiaptomus numidicus), from a reference site. As target zones, for neutral markers, were selected ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) were selected, both consisting of alternating of conserved and variable sequences, conducive to polymorphisms’ occurrence and, therefore, more likely to find “contaminant indicative bands” by PCR-RFLP. A lethal test was conducted with a single concentration of copper (0.5mgL-1) and, using a“time-to-dead assay”, different categories of tolerance were found. The LT50value was 28.5 hours, with a confidence interval from 23.8 to 34.2 hours. The results of PCR-RFLP did not allow the distinction between sensitive organisms (those who died until 2 hours of exposure) and tolerant organisms (those who survived until 64 hours of exposure), because the bands’ patterns of DNA fragments were the same for both groups.Second, the mutagenic potential of acid mine drainage effluent (from de São Domingos mine, Portugal) was evaluated, to test if contaminant-induced mutations could occur at pertinent concentrations, overestimating the genetic diversity evaluated through neutral markers. It was used the Allium cepa test of cytotoxicity and genotoxicity. Mutagenicity was determined by the observation of micronuclei and chromosomal breaks in meristematic cells and was registered at the lowest AMD concentrations used (0.1 and 1% AMD). The mutagenic effect of the AMD persisted after the cessation of exposure (24 and 48 hours recovery tests). Those results indicated that the mutagenic potential of AMD may have contributed to the increased genetic variability of an impacted population of D. longispina reported by Martins et al. (2009) and Silva et al. (2010). Third, the quantitative PCR (q-PCR) technique was used to profile D. magna gene expression, over 168 hours, corresponding to the growth from neonate up to the first reproductive stage (eggs in the brood pouch). A first approach to determin the baseline variability of target genes (Mt, Fer, COI, ND2, LDH, IMP, Vtg, and EcR) and of the reference genes (Act, GAPDH and UbC) was made, by comparing the obtained profiles with physiological processes during growth.Most of the genes had an increased expression at 72 and 144 hours, which corresponds to eggs provisioning at 1st and 2nd brood, meaning that ovaries maturation is a complex prossess involving various genes and metabolic pathways. The sharp decrease that occurs at 168 hours revealed the influence of the embryonic development process, and consequently, for a later analysis, eggs or embryos inside the brood pouch should be removed prior to RNA extraction. The genes of glicolysis and electron transport pathway (COI, ND2 and LDH) showed a higer basal variability and over extended 95% confidence intervals. No relation was found with the EcR gene expression pattern and ecdysis periods during D. magna growth, revealing that this EcR isoforme should have a preponderant role in embryonic development.The normalization process may be responsible for diferences at gene expression profiles. The metal transporters group (Mt and Fer) showed more significant results when normalized with geNorm than with BeastKeeper methodologies. The IMP was the gene most affected by copper exposure, being up-regulated at 6, 24, 48, 72, and 168 hours, with a down-regulation at 96 hours of exposure. Despide the inositol relation with growth processes, it also may act as a defense against copper oxidative damage.