Efeito de fatores ambientais na longevidade de Daphnia magna

Mestrado em Biologia Molecular e CelularLongevidade é a capacidade de um organismo sobreviver para além da idade média de morte para a espécie a que pertence. Pode ser modulada por vários parâmetros ambientais que influenciam o metabolismo celular, a oxidação, ou a integridade do ADN. Até ao presente já foram realizados alguns estudos com o objetivo de compreender a influência de vários fatores ambientais na longevidade dos organismos. No entanto, a maior parte destes estudos são limitados a um número reduzido de espécies modelo. Deste modo, é pertinente gerar mais conhecimento sobre a estrutura do genoma e respostas funcionais de genes às condições ambientais em espécies com ecologias bem conhecidas, de modo a promover uma melhor compreensão das interações gene-ambiente num contexto evolutivo. A metodologia mais direta para abordar esta questão é identificar genes ou intervenções que funcionam de forma semelhante de modo a se poder proceder à modulação do tempo de vida nos organismos. Componentes de insulina ou o fator de crescimento 1 da família da insulina (IGF- 1), a cínase da rapamicina (TOR) e as sirtuínas (SIR) da família das desacetilases, reguladas pelo gene PNC1 (pirazinamidase e nicotinamidase), são alguns exemplos que apresentam essa característica. Levando isso em consideração, o principal objetivo deste trabalho foi avaliar a influência de diversos parâmetros abióticos na longevidade de duas linhagens clonais da espécie Daphnia magna Straus (K6 e BEAK), da sub ordem Cladocera. Para atingir este objectivo foram avaliados os efeitos da temperatura, da restrição alimentar e de diferentes níveis de cobre na reprodução (número total de neonatos produzidos) e no tempo de vida de D. magna. No caso de restrição calórica e cobre, foram ainda avaliados os efeitos na expressão génica e integridade (ocorrência de mutações) do gene PNC1, respetivamente. Daphnia magna foi selecionada como organismo para este estudo, por apresentar uma ecologia bem documentada, ser de fácil manutenção em laboratório e por se reproduzir por partenogénese, o que permite o estabelecimento de linhagens clonais, sendo desta forma uma ferramenta para discriminar diferenças genéticas (entre clones) do background experimental. Além disso, o genoma da espécie Daphnia pulex foi já sequenciado, permitindo a avaliação da relação entre a estrutura, expressão génica e respostas a nível populacional às alterações ambientais. De um modo geral, os resultados obtidos revelaram uma dependência entre linhagens clonais e a influência dos parâmetros ambientais na longevidade. O aumento da concentração de cobre e da temperatura provocou uma redução na produção de descendência e também na longevidade de D. magna. A restrição alimentar também induziu um decréscimo na produção de descendência nas duas linhagens clonais. A análise das sequências de ADN apenas revelou polimorfismos semelhantes em todas as amostras, o que sugere que não foram induzidas quaisquer mutações devido a exposição aos fatores ambientais em estudo. Também não foram identificados efeitos significativos de restrição calórica na expressão relativa do gene PNC1.Longevity is the ability to survive beyond the average age of death for the species. It can be modulated by several environmental parameters that influence cellular metabolism, oxidation, or DNA integrity. A set of works has already been carried out in order to understand the influence of environmental factors in longevity. However, most of these studies are restricted to a narrow number of model species. It is then pertinent to generate more knowledge on the genome structure and on the functional responses of genes to environmental conditions within species with tractable ecologies, aiming to improve the understanding of gene-environment interactions in an evolutionary context. The most direct way to address this problem is to identify genes or interventions that function similarly to modulate life span in different organisms. Components of the insulin or insulin-like growth factor 1 (IGF-1) signaling pathway, the nutrient-responsive target of rapamycin (TOR) kinase and the sirtuin (SIR) family of protein deacetylases, regulated by PNC1 gene (pyrazinamidase and nicotinamidase), among others, have been found to have this property. Taking this into consideration, the main goal of this work was to assess the influence of several environmental parameters in the longevity of two clonal lineages of the cladoceran species Daphnia magna Straus (K6 and BEAK). To attain this purpose, the effects of temperature, food restriction and different copper levels were evaluated in the reproduction (total number of neonates produced) and life span of D. magna. The effects in the gene expression and genomic integrity of the PNC1 gene (occurrence of mutations) were assessed in the food restriction and copper assays, respectively. The model organism Daphnia magna was selected to address the purposed objective as its ecology is well understood, it is of easy maintenance and reproduces by parthenogenesis, which allows the creation of clonal lineages, thus providing a tool to discriminate genetically based differences (among clones) from the experimental background. In addition, the genome of the closely related Daphnia pulex has been sequenced, allowing the assessment of the relationship between structure, gene expression and population-level responses to environmental changes. In general, the obtained results revealed a clonal lineage dependency on the influence of the environmental parameters in longevity. An increased temperature and copper concentration provoked a reduction in the reproductive output and as well as in the longevity of daphnids. Food restriction also induced a decrease in the reproductive output of both clonal lineages of D. magna. After DNA sequencing, only similar SNPs were found in all samples and therefore, no mutational alterations were caused by exposure to the studied environmental factors. No significant effects occurred in the relative gene expression of PNC1 due to caloric restriction

Insights into the restoration of tributyltin contaminated environments using marine bacteria from Portuguese fishing ports

Tributyltin (TBT) is an organotin chemical mainly used as biocide in marine antifouling paints. Despite the restrictions and prohibitions on its use, TBT is still an environmental problem due to its extensive application and subsequent release into the environment, being regarded as one of the most toxic chemicals released into the marine ecosystems. Microorganisms inhabiting impacted sites are crucial for their restoration since they have developed mechanisms to tolerate and break down pollutants. Nonetheless, transformation products resulting from the degradation process may still be toxic or, sometimes, even more toxic than the parent compound. The determination of the parent and degradation products by analytical methods, although necessary, may not be ecologically relevant since no information is provided regarding their ecotoxicity. In this study, marine bacteria collected from seven Portuguese fishing ports were isolated and grown in the presence of TBT. Bacteria that exhibited higher growth were used to bioremediate TBT-contaminated waters. The potential of these bacteria as bioremediation agents was evaluated through ecotoxicological assays using the sea snail Gibbula umbilicalis as model organism. Data suggested that some TBT-tolerant bacteria, such as Pseudomonas putida, can reduce the toxicity of TBT contaminated environments. This work contributes to the knowledge of TBT-degrading bacteria.info:eu-repo/semantics/publishedVersio

Impact of anthropogenic noise on the survival and development of meagre (Argyrosomus regius) early life stages

The growth of human populations has been driving an unprecedent and widespread increase in marine traffic, posing a real threat to marine biodiversity. Even though we are now aware of the negative effects of shipping noise exposure on fish, information about the impact on their early life stages continues to lack. Meagre (Argyrosomus regius) is a vocal fish that uses estuaries with high levels of anthropogenic noise pollution as both breeding areas and nurseries. Here, the effects of boat noise exposure on the development and survival of meagre larvae were studied. Embryos and larvae were exposed to either noise (boat noise playback) or control treatments (coils producing a similar electric field to the speakers) and hatching rate, survival rate, morphometric traits and stress-related biomarkers, at hatching and at 2 days-post-hatching (dph) were analyzed. Results showed no conclusive effects of the impact of boat noise playback, even though there was an increased lipid droplet consumption and a decrease in body depth at 2dph larvae under this stressor. The assessment of oxidative stress and energy metabolism-related biomarkers at hatching showed a marginal decrease in superoxide dismutase (SOD) activity and no changes in DNA damage or electron transport system activity (ETS), although it cannot be disregarded that those effects could only be visible at later stages of larval development. Whether these morphological and developmental results have implications in later stages remains to be investigated. Further studies with longer exposure and wild meagre could help deepen this knowledge and provide a better understanding of how anthropogenic noise can impact meagre early stagesFundação para a Ciência e Tecnologia - FCTinfo:eu-repo/semantics/publishedVersio

Effect Biomarkers of the Widespread Antimicrobial Triclosan in a Marine Model Diatom

The present-day COVID-19 pandemic has led to the increasing daily use of antimicrobials worldwide. Triclosan is a manmade disinfectant chemical used in several consumer healthcare products, and thus frequently detected in surface waters. In the present work, we aimed to evaluate the effect of triclosan on diatom cell photophysiology, fatty acid profiles, and oxidative stress biomarkers, using the diatom Phaeodactylum tricornutum as a model organism. Several photochemical effects were observed, such as the lower ability of the photosystems to efficiently trap light energy. A severe depletion of fucoxanthin under triclosan application was also evident, pointing to potential use of carotenoid as reactive oxygen species scavengers. It was also observed an evident favouring of the peroxidase activity to detriment of the SOD activity, indicating that superoxide anion is not efficiently metabolized. High triclosan exposure induced high cellular energy allocation, directly linked with an increase in the energy assigned to vital functions, enabling cells to maintain the growth rates upon triclosan exposure. Oxidative stress traits were found to be the most efficient biomarkers as promising tools for triclosan ecotoxicological assessments. Overall, the increasing use of triclosan will lead to significant effects on the diatom photochemical and oxidative stress levels, compromising key roles of diatoms in the marine system.info:eu-repo/semantics/publishedVersio

Anoctamin 5: A New Candidate Gene For Portuguese Patients With Adult Onset Limb-Girdle Muscular Dystrophy

Introduction: The limb-girdle muscular dystrophies (LGMDs) show wide genetic and clinical heterogeneity. Recessive mutations in the ANO5 gene, which encodes a putative calcium-activated chloride channel of the anoctamin family, have been recently identified in families with LGMD type 2L and non-dysferlin distal muscular dystrophy (MMD3). The LGMD2L phenotype is characterized by proximal weakness, with prominent asymmetrical quadriceps femoris and biceps brachii atrophy, whereas MMD3 is associated with distal weakness, particularly of calf muscles. Methods: In a group of 125 patients with clinical LGMD, but no mutations in other candidate genes involved in LGMD, we screened the “common” mutation c.191dupA. Subsequently, in 10 selected patients the entire coding region of ANO5 was fully sequenced. Results: Mutations were identified in 4 patients (3 families), all presenting hyperCKemia and adult onset proximal lower limb weakness. The common mutation c.191dupA was found in one family (2 patients), in a homozygous state. This mutation results in a frameshift with a consequent premature stop codon (p.Asn64LysfsX15), triggering nonsense-mediated mRNA decay. A novel substitution identified in exon 18 (c.2012A>G), predictably a missense mutation, was shown to in fact create a new donor splice site. mRNA studies confirmed aberrant splicing in exon 18, promoting an in-frame deletion of 18 nucleotides (r.2012_2029del) that results in a truncated protein (p.Tyr671_Val677delinsPhe). The third patient had a heterozygous nucleotide substitution in exon 8, c.692G>T, predicted to result in a missense mutation (p.Gly231Val). The Gly231 residue, localized in the N-terminal domain, is evolutionarily conserved. In this patient the second mutation has not yet been identified. Conclusion: Although c.191dupA was detected in only 1/125 patients, systematic sequencing of ANO5 in 10 patients revealed a further two positive cases, indicating that the anoctaminopathies may account for a reasonable number of our LGMD patients

Scyliorhinus canicula (Linnaeus, 1758) metal accumulation: A public health concern for Atlantic fish consumers?

One of the most landed sharks in Portuguese fisheries is the lesser-spotted dogfish (Scyliorhinus canicula), which is ever-present in Portuguese fish markets and consumed as cheap fish protein source. The focus of this study was to evaluate element contamination in consumed tissues of Atlantic S. canicula, with the intent of safeguarding possible public health issues. A total of 74 specimens were analysed for metals and metalloids in the deeper white muscle and skin. Arsenic, zinc, iron and aluminium were the elements with higher mean values. There was a tendency for higher levels in the skin, with differences between life-stage and gender. Many individuals surpassed stipulated guideline limits for mercury and arsenic, posing a risk for human consumption (according to the health risk assessment performed for the average Portuguese fish consumption) or even for use in feed production. Besides the public health concern, this study also evidences troubling signs on marine contamination status.info:eu-repo/semantics/publishedVersio

Assessment of contaminants in blue sharks from the Northeast Atlantic: Profiles, accumulation dynamics, and risks for human consumers

This study had the support of Fundação para a Ciência e a Tecnologia (FCT) through the project BLUESHARKER (PTDC/CTAAMB/29136/2017), co-financed by COMPETE2020 (POCI-01-0145-FEDER-029136), the grant to MARE – Marine and Environmental Sciences Centre (UIDB/04292/2020 and UIDP/04292/2020) and the Associate Laboratory ARNET (LA/P/0069/2020), the grant awarded to Luís Alves (SFRH/BD/122082/2016), and the transitory norm contract to Filipe Ceia (DL57/2016/CP1370/CT90). Sara Novais is funded by national funds (OE), through FCT, in the scope of the framework contract foreseen in the numbers 4, 5, and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19.Chemical pollution is a major threat to marine ecosystems, and top predators such as most shark species are extremely vulnerable to being exposed and accumulating contaminants such as metals and persistent organic pollutants (POPs). This work aimed to study the degree, composition, and the sources of contamination in the blue shark (Prionace glauca) inhabiting the Northeast Atlantic, as well as the potential risk faced by human consumers. A total of 60 sharks were sampled in situ aboard fishing vessels, and the concentrations of a set of metals and POPs were analysed in various tissues and complemented with stable isotope analyses. High levels of contaminants were found in most sharks sampled. The concentrations of most metals were higher in the muscle when compared with the liver. Regarding the dangers to consumers posed by the concentrations of arsenic (As), mercury (Hg), and lead (Pb), over 75% of the sharks presented muscle concentrations of at least one contaminant above the legal limits for human consumption, and a risk assessment determined that consumption of meat of these sharks exceeding 0.07 Kg per week could potentially expose human consumers to dangerous amounts of methylmercury (MeHg). Additionally, the assessment of single contaminants may lead to an underestimation of the risk for the human health. Finally, the overall accumulation of contaminants seems to be mostly influenced by the sharks’ geographical distribution, rather than sex, size, or trophic level of their prey.info:eu-repo/semantics/publishedVersio

Are tolerance processes limiting the responses of Hediste diversicolor to cadmium exposure? A multimarker approach

This study had the support of Fundação para a Ciência e a Tecnologia (FCT) through the grant awarded to Carmen A. Pedro (SFRH/BD/124200/2016) and through the strategic projects UIDB/04292/2020 and UIDP/04292/2020 granted to MARE-Marine and Environmental Sciences Centre and the project LA/P/0069/2020 granted to the Associate Laboratory ARNET. Sara Novais is funded by national funds (OE), through FCT – Fundação para a Ciência e a Tecnologia, I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19.Cadmium (Cd) is considered a priority hazardous substance under the European Community Directive 2013/39 due to its ecotoxicity. The ragworm Hediste diversicolor (O.F. Müller, 1776), a common species in estuaries and coastal lagoons, plays an important ecological role in these ecosystems and is a suitable bioindicator of environmental chemical contamination. In this study, H. diversicolor was chosen as an ecotoxicological model with the aim of evaluating the responses to Cd contamination, considering a multi-biomarker approach (mortality, biometry, behaviour, Cd bioaccumulation, oxidative stress and damage, and energy metabolism). Also, the hypothesis of different tolerances resulting in different responses was evaluated, by collecting worms from three systems distinctly impacted by metal contamination (Mondego estuary, Óbidos Lagoon and Sado estuary – Portugal). Animals were exposed under laboratory conditions to cadmium (10, 50 and 100 μg/L), for 10 days. Significant differences were observed in responses amongst worms originating from the different sites. Organisms from the less impacted systems revealed greater effects on mortality, biomass decrease and burrowing behaviour, as well as higher bioaccumulation potential, after exposure to Cd. Biochemical and behaviour impairments were observed as a consequence of Cd exposure, although not in a concentration-dependant manner. The results obtained in this study reinforce the importance of integrating endpoint responses, at the individual and subindividual levels, to assess potential changes induced by pollutants in the physiological status and fitness of H. diversicolor and help to predict what their ecological consequences might be.info:eu-repo/semantics/publishedVersio

Assessment of contaminants in blue sharks from the Northeast Atlantic: Profiles, accumulation dynamics, and risks for human consumers

Chemical pollution is a major threat to marine ecosystems, and top predators such as most shark species are extremely vulnerable to being exposed and accumulating contaminants such as metals and persistent organic pollutants (POPs). This work aimed to study the degree, composition, and the sources of contamination in the blue shark (Prionace glauca) inhabiting the Northeast Atlantic, as well as the potential risk faced by human consumers. A total of 60 sharks were sampled in situ aboard fishing vessels, and the concentrations of a set of metals and POPs were analysed in various tissues and complemented with stable isotope analyses. High levels of contaminants were found in most sharks sampled. The concentrations of most metals were higher in the muscle when compared with the liver. Regarding the dangers to consumers posed by the concentrations of arsenic (As), mercury (Hg), and lead (Pb), over 75% of the sharks presented muscle concentrations of at least one contaminant above the legal limits for human consumption, and a risk assessment determined that consumption of meat of these sharks exceeding 0.07 Kg per week could potentially expose human consumers to dangerous amounts of methylmercury (MeHg). Additionally, the assessment of single contaminants may lead to an underestimation of the risk for the human health. Finally, the overall accumulation of contaminants seems to be mostly influenced by the sharks’ geographical distribution, rather than sex, size, or trophic level of their prey.This study had the support of Fundaçao ˜ para a Ciˆencia e a Tecnologia (FCT) through the project BLUESHARKER (PTDC/CTAAMB/29136/ 2017), co-financed by COMPETE2020 (POCI-01-0145-FEDER-029136), the grant to MARE – Marine and Environmental Sciences Centre (UIDB/ 04292/2020 and UIDP/04292/2020) and the Associate Laboratory ARNET (LA/P/0069/2020), the grant awarded to Luís Alves (SFRH/BD/ 122082/2016), and the transitory norm contract to Filipe Ceia (DL57/ 2016/CP1370/CT90). Sara Novais is funded by national funds (OE), through FCT, in the scope of the framework contract foreseen in the numbers 4, 5, and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 1

Evidence of contamination-associated damage in blue sharks (Prionace glauca) from the Northeast Atlantic

This study had the support of Fundação para a Ciência e a Tecnologia (FCT) through the project BUESHARKER (PTDC/CTAAMB/29136/2017), co-financed by COMPETE2020 (POCI-01-0145-FEDER-029136), the Strategic Project granted to MARE – Marine and Environmental Sciences Centre (UIDB/04292/2020 and UIDP/04292/2020), the project granted to the Associate Laboratory ARNET (LA/P/0069/2020), and the grant awarded to Luís Alves (SFRH/BD/122082/2016). Vanessa F. Fonseca was supported by FCT, via research contract 2021.00244.CEECIND. Sara Novais is funded by national funds (OE), through FCT, in the scope of the framework contract foreseen in the numbers 4, 5, and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19.Top predators such as most shark species are extremely vulnerable to amassing high concentrations of contaminants, but not much is known about the effects that the contaminant body burden imparts on these animals. Species like the blue shark (Prionace glauca) are very relevant in this regard, as they have high ecological and socioeconomic value, and have the potential to act as bioindicators of pollution. This work aimed to assess if differences in contaminant body burden found in blue sharks fromthe Northeast Atlantic would translate into differences in stress responses. Biochemical responses related to detoxification and oxidative stress, and histological alterations were assessed in the liver and gills of 60 blue sharks previously found to have zone-related contamination differences. Similar zone-related differences were found in biomarker responses, with the sharks from the most contaminated zone exhibiting more pronounced responses. Additionally, strong positive correlations were found between contaminants (i.e., As, PCBs, and PBDEs) and relevant biomarkers (e.g., damaged DNA and protective histological alterations). The present results are indicative of the potential that this species and these tools have to be used to monitor pollution in different areas of the Atlantic.info:eu-repo/semantics/publishedVersio