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

Tese de doutoramento em Biociências apresentada à Faculdade de Ciências e Tecnologia da Universidade de CoimbraGenetic 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

Could contaminant induced mutations lead to a genetic diversity overestimation?

Contaminant driven genetic erosion reported through the inspection of selectable traits can be underestimated using neutral markers. This divergence was previously reported in the aquatic system of an abandoned pyrite mine. The most sensitive genotypes of the microcrustacean cladoceran Daphnia longispina were found to be lacking in the impacted reservoir near the entrance of the metal rich acid mine drainage (AMD). Since that divergence could be, at least partially, accounted for by mutagenicity and genotoxicity of the AMD, the present study aimed at providing such a characterization. The Allium cepa chromosomal aberration assay, using root meristematic cells, was carried out, by exposing seeds to 100, 10, 1, and 0.1 % of the local AMD. Chromosomal aberrations, cell division phases and cell death were quantified after the AMD exposure and after 24 and 48 h recovery periods. The AMD revealed to be mutagenic and genotoxic, even after diluting it to 1 and 0.1 %. Dilutions within this range were previously found to be below the lethality threshold and to elicit sublethal effects on reproduction of locally collected D. longispina clonal lineages Significant mutagenic effects (micronuclei and chromosomal breaks) were also found at 0.1 % AMD, supporting that exposure may induce permanent genetic alterations. Recovery tests showed that AMD genotoxic effects persisted after the exposure.This study was partially was funded by FSE and POPH by national funds through Fundação para a Ciência e a Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior (Ph.D. Grant SFRH/BD/28526/2006 to Olímpia Sobral)

Impacto de um modelo de supervisão clínica nas capacidades da inteligência emocional em enfermeiros

Objective: to analyze the impact of the implementation of a model of clinical supervision over the emotional intelligence capacities of nurses. Methods: mixed methods study. Quantitative data were collected before and after the implementation of the Model, using the scale of Emotional Intelligence Capacities of Nurses; analyses were carried out using descriptive statistics and paired samples were analyzed using the t-test. 47 nurses from one hospital participated. Qualitative data were obtained from the analysis of the discourses of the nurses in a public presentation, using thematic/ categorizing content analysis. The findings were integrated at the end to generate the results. Results: no statistically significant differences were found with the implementation of the Model. However, the nurses involved recognized the positive impact of the SafeCare Model in the development of emotional competences. Conclusion: the study contributed for clinical supervision and for the development of emotional competencesObjetivo: analisar o impacto da implementação de um modelo de supervisão clínica em enfermagem nas capacidades da inteligência emocional dos enfermeiros. Métodos: estudo de métodos mistos. Colheu-se os dados quantitativos antes e após a implementação do Modelo, pela escala Capacidades da Inteligência Emocional em Enfermeiros; analisou- se por estatística descritiva e pelo teste-t para amostras emparelhadas. Participaram 47 enfermeiros de um hospital. Obteve-se os dados qualitativos pela análise dos discursos dos enfermeiros numa apresentação pública, através de técnicas de análise de conteúdo tipo temática/categorial. Os achados foram integrados no final para gerar os resultados. Resultados: não se verificou diferenças estatisticamente significativas com a implementação do Modelo. Contudo, os enfermeiros envolvidos reconheceram o impacto positivo do Modelo SafeCare no desenvolvimento das competências emocionais. Conclusão: o estudo apresentou contributos para a supervisão clínica e para o desenvolvimento das competências emocionais.info:eu-repo/semantics/publishedVersio

Biodiversity of leaf litter fungi in streams along a latitudinal gradient

Global patterns of biodiversity have emerged for soil microorganisms, plants and animals, and the extraordinary significance of microbial functions in ecosystems is also well established. Virtually unknown, however, are large- scale patterns of microbial diversity in freshwaters, although these aquatic ecosystems are hotspots of biodiversity and biogeochemical processes. Here we report on the first large-scale study of biodiversity of leaf-litter fungi in streams along a latitudinal gradient unravelled by Illumina sequencing. The study is based on fungal commu- nities colonizing standardized plant litter in 19 globally distributed stream locations between 69°N and 44°S. Fungal richness suggests a hump-shaped distribution along the latitudinal gradient. Strikingly, community com- position of fungi was more clearly related to thermal preferences than to biogeography. Our results suggest that identifying differences in key environmental drivers, such as temperature, among taxa and ecosystem types is critical to unravel the global patterns of aquatic fungal diversity

Atividades de enzimas relacionadas ao estresse oxidativo e membranas celulares em um macroinvertebrado fragmentador exposto a urânio

Rivers are prone to contamination from agricultural activities, industrial waste and mining, with effects on the biota ranging from the scale of biochemical processes to that of ecosystems. Ongoing climate change requires the replacement of carbon energy sources with alternative energies, and nuclear power is an option. Uranium mining may result in run-off and the contamination of water courses. We investigated the effects of uranium on enzyme activities (cholinesterases (ChEs), Na+K+-ATPase, glutathione S-transferase (GST) and catalase (CAT)) in a freshwater caddisfly shredder Schizopelex festiva exposed to six U concentrations up to 100 μg/l. Enzyme activities and U accumulation were determined at 24 h and 32 d of exposure. We also measured growth rates (32 d) and calculated bioconcentration factors, as the ratio of U in the whole body of the invertebrates to U concentration in the test water. Enzyme activity from 24 h to 32 d was reduced for ChEs (~52 %), GST (~44 %) and CAT (78 %). No change was observed for Na+K+ATPase activities. Enzyme activities for Na+K+ATPase decreased after 32 d only at the highest U concentration. For GST activities, there were significant differences among concentrations for each time, but the treatments did not differ from the control groups. The growth rates of S. festiva under laboratory conditions averaged 6.23 ± 0.66 μg g-1 animal d-1, with no significant differences between treatments and control groups. The ratio of U in the invertebrate body to U concentrations in the water ranged from 211 to 1663, increasing with time and decreasing with the concentration of U in the water. The exposure of S. festiva to U resulted in its accumulation and triggered changes in the activities of some enzymes, but the species was in general tolerant to realistic values observed in the field. If our findings can be extrapolated to other consumers, then larger amounts of U would be needed to cause measurable biological changes.Os rios são propensos a receber poluentes provenientes das atividades agrícolas, industriais e de mineração, com efeitos na biota que vão desde processos bioquímicos até os ecossistemas. A substituição das fontes de energia do carbono por energias alternativas faz com que a energia nuclear possa ser uma opção frente às alterações climáticas. A mineração do urânio pode resultar no escoamento de metais e contaminação das águas superficiais. Os efeitos do urânio nas atividades enzimáticas das colinesterases (ChEs), Na+K+-ATPase, glutationa S-transferase (GST) e catalase (CAT) foram investigados, usando como organismo-teste o tricóptero Schizopelex festiva, expostos a seis concentrações de até 100 μg/l. Fatores de bioconcentração (relação entre U nos invertebrados e U na água) e taxas de crescimento também foram calculados. As atividades das enzimas e a acumulação de U foram determinadas em 24 h e 32 d. As atividades enzimáticas foram reduzidas de 24 h para 32 d para ChEs (~52 %), GST (~44 %) e CAT (~78 %), enquanto que para a Na+K+ATPase não se observaram diferenças entre os tempos de exposição. As atividades enzimáticas da Na+K+ATPase reduziram após 32 d, mas só na mais alta concentração de U. Para GST, houve diferenças nas atividades enzimáticas entre concentrações de U entre períodos de exposição, mas não se observaram diferenças entre controle (0 μg/l) e as restantes concentrações. As taxas de crescimento de S. festiva foram em média de 6.23 ± 0.66 μg g-1 animal d–1, sem diferenças significativas entre tratamentos. Os fatores de bioconcentração variaram entre 211 e 1663, aumentando com o tempo de exposição e diminuindo com a concentração de U na água. A exposição de S. festiva ao U resultou na sua acumulação e provocou alterações nas atividades de algumas enzimas, mas esta espécie foi em geral tolerante a valores encontrados nas águas superficiais. Se nossos resultados puderem ser estendidos a outros consumidores, quantidades de U superiores às testadas seriam necessárias para causar alterações biológicas mensuráveis

Organic-matter decomposition as a bioassessment tool of stream functioning: A comparison of eight decomposition-based indicators exposed to different environmental changes

International audienceOrganic-matter decomposition has long been proposed as a tool to assess stream functional integrity, but this indicator largely depends on organic-matter selection. We assessed eight decomposition-based indicators along two well-known environmental gradients, a nutrient-enrichment gradient (0.2-1.4 mg DIN/L) in central Portugal and an acidification gradient (pH: 4.69-7.33) in north-eastern France to identify the most effective organicmatter indicator for assessing stream functional integrity. Functional indicators included natural leaf litter (alder and oak) in 10-mm and 0.5-mm mesh bags, commercial tea (Lipton green and rooibos teas in 0.25-mm mesh bags), wood sticks (wood tongue depressors) and cotton strips. Biotic indices based on benthic macroinvertebrates (IPtIN for Portugal and IBGN for France) were calculated to compare the effectiveness of structural and functional indicators in detecting stream impairment and to assess the relationship between both types of indicators. The effectiveness of organic-matter decomposition rates as a functional indicator depended on the stressor considered and the substrate used. Decomposition rates generally identified nutrient enrichment and acidification in the most acidic streams. Decomposition rates of alder and oak leaves in coarse-mesh bags, green and rooibos teas and wood sticks were positively related with pH. Only decomposition rates of rooibos tea and wood sticks were related with DIN concentration; decomposition rates along the nutrient-enrichment gradient were confounded by differences in shredder abundance and temperature among streams. Stream structural integrity was good to excellent across streams; the IPtI N index was unrelated to DIN concentration, while the IBGN index was positively related with pH. The relationships between decomposition rates and biotic indices were loose in most cases, and only decomposition rates of alder leaves in coarse-mesh bags and green tea were positively related with the IBGN. Commercial substrates may be a good alternative to leaf litter to assess stream functional integrity, especially in the case of nutrient enrichment

In Vitro Development of Parthenogenetic Eggs: A Fast Ecotoxicity Test with Daphnia magna?

In the present study, the authors investigated and defined development stages of Daphnia magna eggs that could be potential endpoints for sublethal toxicity tests with in vitro parthenogenetic egg cultures. Such an "egg test" could be a cost-effective alternative to the classic 21-day test with D. magna. Three main stages of embryonic development were considered: release of the egg external membrane, release of the internal membrane, and separation of the caudal spine. The first embryonic stage was attained approximately 30 h after transfer of eggs from ovaries to the brood chamber (considered as time zero), the second stage at 48 h, and the third stage at 68 h. Embryonic development was considered completed with the caudal spine separation. Thereafter, juveniles were able to swim in the water column. Egg mortality, duration of each egg stage, egg diameter, and egg abnormalities were investigated as potential endpoints. In vitro tests were carried out with several toxicants (DBS, 3,4-DCA, cadmium, and copper) and with acid mine drainage, sensitivity generally being higher than with the accepted chronic 21-day test with D. magna.http://www.sciencedirect.com/science/article/B6WDM-458W5FX-2Y/1/bef7274e1fa0fddfbb9a8ddf987efad

Impacto de um modelo de supervisão clínica nas capacidades da inteligência emocional em enfermeiros

Objective: to analyze the impact of the implementation of a model of clinical supervision over the emotional intelligence capacities of nurses. Methods: mixed methods study. Quantitative data were collected before and after the implementation of the Model, using the scale of Emotional Intelligence Capacities of Nurses; analyses were carried out using descriptive statistics and paired samples were analyzed using the t-test. 47 nurses from one hospital participated. Qualitative data were obtained from the analysis of the discourses of the nurses in a public presentation, using thematic/categorizing content analysis. The findings were integrated at the end to generate the results. Results: no statistically significant differences were found with the implementation of the Model. However, the nurses involved recognized the positive impact of the SafeCare Model in the development of emotional competences. Conclusion: the study contributed for clinical supervision and for the development of emotional competences.Objetivo: analisar o impacto da implementação de um modelo de supervisão clínica em enfermagem nas capacidades da inteligência emocional dos enfermeiros. Métodos: estudo de métodos mistos. Colheu-se os dados quantitativos antes e após a implementação do Modelo, pela escala Capacidades da Inteligência Emocional em Enfermeiros; analisou-se por estatística descritiva e pelo teste-t para amostras emparelhadas. Participaram 47 enfermeiros de um hospital. Obteve-se os dados qualitativos pela análise dos discursos dos enfermeiros numa apresentação pública, através de técnicas de análise de conteúdo tipo temática/categorial. Os achados foram integrados no final para gerar os resultados. Resultados: não se verificou diferenças estatisticamente significativas com a implementação do Modelo. Contudo, os enfermeiros envolvidos reconheceram o impacto positivo do Modelo SafeCare no desenvolvimento das competências emocionais. Conclusão: o estudo apresentou contributos para a supervisão clínica e para o desenvolvimento das competências emocionais

Biodiversity of leaf litter fungi in streams along a latitudinal gradient

Global patterns of biodiversity have emerged for soil microorganisms, plants and animals, and the extraordinary significance of microbial functions in ecosystems is also well established. Virtually unknown, however, are largescale patterns of microbial diversity in freshwaters, although these aquatic ecosystems are hotspots of biodiversity and biogeochemical processes. Here we report on the first large-scale study of biodiversity of leaf-litter fungi in streams along a latitudinal gradient unravelled by Illumina sequencing. The study is based on fungal communities colonizing standardized plant litter in 19 globally distributed stream locations between 69°N and 44°S. Fungal richness suggests a hump-shaped distribution along the latitudinal gradient. Strikingly, community composition of fungi was more clearly related to thermal preferences than to biogeography. Our results suggest that identifying differences in key environmental drivers, such as temperature, among taxa and ecosystem types is critical to unravel the global patterns of aquatic fungal diversity3F10-AC72-52D0 | Verónica Ferreirainfo:eu-repo/semantics/publishedVersio