Development of an ecotoxicological protocol for the deep-sea fauna using the hydrothermal vent shrimp Rimicaris exoculata

In light of deep-sea mining industry development, particularly interested in massive-sulphide deposits enriched in metals with high commercial value, efforts are increasing to better understand potential environmental impacts to local fauna. The aim of this study was to assess the natural background levels of biomarkers in the hydrothermal vent shrimp Rimicaris exoculata and their responses to copper exposure at in situ pressure (30MPa) as well as the effects of depressurization and pressurization of the high-pressure aquarium IPOCAMP. R. exoculata were collected from the chimney walls of the hydrothermal vent site TAG (Mid Atlantic Ridge) at 3630m depth during the BICOSE cruise in 2014. Tissue metal accumulation was quantified in different tissues (gills, hepatopancreas and muscle) and a battery of biomarkers was measured: metal exposure (metallothioneins), oxidative stress (catalase, superoxide dismutase, glutathione-S-transferase and glutathione peroxidase) and oxidative damage (lipid peroxidation). Data show a higher concentration of Cu in the hepatopancreas and a slight increase in the gills after incubations (for both exposed groups). Significant induction of metallothioneins was observed in the gills of shrimps exposed to 4μM of Cu compared to the control group. Moreover, activities of enzymes were detected for the in situ group, showing a background protection against metal toxicity. Results suggest that the proposed method, including a physiologically critical step of pressurizing and depressurizing the test chamber to enable the seawater exchange during exposure to contaminants, is not affecting metal accumulation and biomarkers response and may prove a useful method to assess toxicity of contaminants in deep-sea species.

Life history & environmental effects on telomere dynamics in Atlantic salmon

While much of the study of molecular biology inevitably focuses on the parts of the genome that contain active genes, there are also non-coding regions that nonetheless play an essential role in maintaining genome integrity. One such region are telomeres, which cap the ends of all eukaryotic chromosomes and play an important role in chromosome protection. Telomere loss occurs at each cell division as a result of the ‘end replication problem’ and a relatively short telomere length is indicative of poor biological state. Thus far, the majority of studies on the dynamics and role of telomeres have been biased towards certain taxa. Research to date has mostly focussed on humans, other mammals and birds. There has been far less research on the telomere dynamics of ectotherms. It is important that we do so, especially since ectothermic vertebrates do not seem to down-regulate telomerase expression in the same way as endotherms, suggesting that their telomere dynamics may be less predictable in the later life stages. The main objective of this thesis was therefore to investigate how life history and environmental effects may influence telomere dynamics in Atlantic salmon Salmo salar. I carried out carefully designed experiments, both in the laboratory and in the wild, using a longitudinal approach where possible, in order to address a number of specific questions that are connected to this central theme. In chapter 2, I demonstrate that there can be significant links between parental life history and offspring telomere dynamics. Maternal life history traits, in particular egg size, were most strongly related to offspring telomere length at the embryonic stages. Paternal life history traits, such as early life growth rate, had a greater association with offspring telomere dynamics in the later stages of development. In chapter 3, using a wild Atlantic salmon population, I found that most individuals experienced a reduction in telomere length during the migratory phase of their life cycle; however the relative rate of telomere loss was dependent on sex, with males experiencing a relatively greater loss. Unexpectedly, I also found that juvenile salmon that had the shortest telomeres at the time of outward migration, had the greatest probability of surviving through to the return migration. In chapter 4, again using a wild system involving experimental manipulations of juvenile Atlantic salmon in Scottish streams, I found that telomere length in juvenile fish was influenced by parental traits and by direct environmental effects. Faster-growing fish had shorter telomeres and there was a greater cost (in terms of reduced telomere length) if the growth occurred in a harsher environment. I also found a positive association between offspring telomere length and the growth history of their fathers (but not mothers), represented by the number of years that fathers had spent at sea. Chapter 5 explored the hypotheses that oxidative DNA damage, catalase (CAT) antioxidant activity and cell proliferation rate are underlying mechanisms linking incubation temperature and telomere dynamics in salmon embryos. No evidence was found for any such effects, but telomere lengths in salmon embryos were found to be significantly affected by the temperature of the water in which they were living. There is also evidence that telomere length significantly increases during embryonic development. In summary, this thesis has shown that a complex mix of environmental and parental effects appear to influence telomere dynamics in Atlantic salmon, with parental effects especially evident during early life stages. It also demonstrated that telomeres lengthen through the embryo stages of development before reducing once the fry begin feeding, indicating that the patterns of telomere loss commonly found in endotherms may differ in ectotherms. Reasons for this variation in telomere dynamics are presented in the final Discussion chapter of the thesis.

Fitoextracção de cádmio de solos contaminados, com plantas seleccionadas

A remediação de locais contaminados com metais pesados usando plantas hiperacumuladoras aparenta ser uma alternativa bastante viável. Neste trabalho comparou-se a acumulação e tolerância ao cádmio (Cd), ambas baseadas nas respostas ao stress oxidativo em três espécies de plantas diferentes: Brassica juncea (L.) Czem., Nicotiana tabacum L. e Solanum nigrum L., descritas na literatura como plantas bastante tolerantes ou até com características híper acumuladoras. As plantas cresceram num solo contaminado com diferentes concentrações de Cd (O- 35 mg kg-1) durante um período de 90 dias. O factor de translocação (FT), utilizado para medir a translocação efectiva do Cd da raiz para a parte aérea, variou consideravelmente entre as espécies desenvolvidas. A N. tabacum foi a planta que apresentou os maiores valores de FT. Neste trabalho foi a única planta que preencheu todas as condições para ser considerada hiperacumuladora para todos os níveis de contaminação do solo. Por outro lado, a S. nigrum apresentou os maiores valores de concentração de Cd nos tecidos, com um FT > 1, na presença de 5 mg Cd kg·1 de solo. Apesar da B. juncea ter apresentado um resultado de FT inferior às restantes, foi a única planta com valores crescentes de FT com o aumento da contaminação de Cd. O stress oxidativo nas plantas desenvolvidas foi avaliado pela peroxidação lipídica e pelas actividades da catalase (CAT), ascorbato peroxidase (APX), guaiacol peroxidase (GPX) e superóxido dismutase (SOO), quer na raiz quer na parte aérea. Foi observado um aumento significativo (versus controlo) na peroxidação lipídica e actividade enzimática da CATe APX na parte aérea da B. juncea, N. tabacum e S. nigrum para os níveis de contaminação mais elevados, 15 e/ou 35 mg Cd kg-1 A B. juncea apresentou maior sensibilidade na resposta da GPX, para todas as concentrações de Cd no solo. A peroxidação lipídica e a actividade da CAT foram superiores na parte aérea em relação à raiz para todas as plantas em todas as contaminações de Cd presentes no solo. A actividade da SOO não apresentou respostas consistentes para nenhuma das plantas. ABSTRACT: Remediation of sites contaminated with heavy metals using hyper accumulators seems a promising alternative to engineering approaches. ln this work, we compared cadmium (Cd) accumulation and tolerance (based on responses to oxidative stress) in three different species, Brassica juncea (L) Czem., Nicotiana tabacum L. and Solanum nigrum L., described in the literature as very tolerant or even as hyper accumulators. The plants were grown in soil spiked with different Cd concentrations (O- 35 mg kg- 1) over a period of 90 days. The translocation factor (TF), used to measure the effectiveness of translocating Cd from roots to shoots, depended greatly on the species. N. tabacum was the plant which exhibited the highest TF values. lt was the only plant under study that fulfilled the conditions of a hyper accumulator for all levels of soil contamination. On the other hand, S. nigrum presented the highest Cd concentration in plant tissues, with TF > 1 in the presence of 5 mg Cd kg-1 of soil. Although B. juncea had presented the lowest TF and Cd concentrations, it was the only plant with TF values increasing with the level of cadmium. Oxidative stress in plants was evaluated by lipid peroxidation and activities of catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and superoxide dismutase (SOO), both in roots and shoots. A significant enhancement (versus control) on lipid peroxidation and enzymatic activity of CAT and APX in shoots of B. juncea, N. tabacum and S. nigrum was observed for the highest levels of Cd in soil, 15 and/or 35 mg Cd kg-1. B. juncea presented the most sensitive response of GPX, for all levels of Cd in soil. Lipid peroxidation and CAT activity were greater in shoots than in roots for all plants and soil Cd concentrations. SOO activity did not present consistent trends for any plant.

he genome and genetics of a high oxidative stress tolerant Serratia sp. LCN16 isolated from the plant parasitic nematode Bursaphelenchus xylophilus

Background: Pine wilt disease (PWD) is a worldwide threat to pine forests, and is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus. Bacteria are known to be associated with PWN and may have an important role in PWD. Serratia sp. LCN16 is a PWN-associated bacterium, highly resistant to oxidative stress in vitro, and which beneficially contributes to the PWN survival under these conditions. Oxidative stress is generated as a part of the basal defense mechanism used by plants to combat pathogenic invasion. Here, we studied the biology of Serratia sp. LCN16 through genome analyses, and further investigated, using reverse genetics, the role of two genes directly involved in the neutralization of H2O2, namely the H2O2 transcriptional factor oxyR; and the H2O2-targeting enzyme, catalase katA. Results: Serratia sp. LCN16 is phylogenetically most closely related to the phytosphere group of Serratia, which includes S. proteamaculans, S. grimessi and S. liquefaciens. Likewise, Serratia sp. LCN16 shares many features with endophytes (plant-associated bacteria), such as genes coding for plant polymer degrading enzymes, iron uptake/ transport, siderophore and phytohormone synthesis, aromatic compound degradation and detoxification enzymes. OxyR and KatA are directly involved in the high tolerance to H2O2 of Serratia sp. LCN16. Under oxidative stress, Serratia sp. LCN16 expresses katA independently of OxyR in contrast with katG which is under positive regulation of OxyR. Serratia sp. LCN16 mutants for oxyR (oxyR::int(614)) and katA (katA::int(808)) were sensitive to H2O2 in relation with wild-type, and both failed to protect the PWN from H2O2-stress exposure. Moreover, both mutants showed different phenotypes in terms of biofilm production and swimming/swarming behaviors. Conclusions: This study provides new insights into the biology of PWN-associated bacteria Serratia sp. LCN16 and its extreme resistance to oxidative stress conditions, encouraging further research on the potential role of this bacterium in interaction with PWN in planta environment.