Embryonic and larval ecology and biochemistry of offshore decapods

Understanding the biology of offshore species is hardened by the difficulties of sampling in the deep-sea environment. Additionally, due to the vastness of the open ocean, knowledge of early life histories of pelagic larvae is still relatively scarce. In decapod species with bentho-pelagic lifestyle, the transition from life in the seafloor to the water column not only is associated with drastic morphological metamorphosis, but also with changes in behavior and feeding ecology. The purpose of the present thesis was to investigate physiological, biochemical and behavioral adaptation occurring during early development of such species. The Norway lobster, Nephrops norvegicus, and the crab Monodaeus couchi were used as a model as these two species are encountered off the NE Atlantic shelf at depth greater than 300 m. Chapter 1 introduces the challenges faced by both adult and larvae inhabiting such remote habitats, including the effect of food availability on development and oceanographic processes on dispersal and recruitment. The thesis follows early life histories, starting with within-brood variability in the fatty acid (FA) profile displayed by developing N. norvegicus embryos. There were no differences in the FA composition of embryos sampled from both sides of the brooding chamber in most females. However, all females exhibited significant differences in the FA profiles of embryos sampled from different pleopods. Potential causes for the variations recorded may be differential female investment during oocyte production or shifts in FA catabolism during the incubation period promoted by embryo’s location within the brooding chamber. Next, feeding rates and digestive enzymes activity of the early stage larvae was investigated in N. norvegicus. Both stages were able to maximize food intake when larvae were scarce and showed increased feeding rate following periods of starvation. Amylase activity indicated that carbohydrates are not the primary energy reserve and that feeding may be required soon after hatching to trigger amylase activity. Protease activity indicated that protein reserves are catabolized under starvation. These results indicate that larvae may maximize prey ingestion in the presence of plankton patches with higher food abundance and minimize the deleterious effects induced by previous periods of intermittent starvation or unsuitable prey densities/types. Additionally, changes in enzymatic activity may allow newly hatched N. norvegicus larvae to metabolize protein reserves to overcome short-term starvation. Vertical migration behavior and the influence of oceanographic properties were studied next. All zoeal stages of M. couchi displayed reverse diel vertical migration. Abundance of early stages was correlated with chlorophyll a levels. An ontogenic shift in vertical distribution explained the results; earlier zoeal stages remain in the food-rich upper water column while later stages migrate to the bottom for settlement. This vertical migration behavior is likely to affect horizontal distribution of larvae. Indeed, global current patterns will result in low inter-annual variations in decapod larvae recruitment, but short term variations such as upwelling events will cause deviation from the expected dispersal pattern. Throughout development, from the embryo to metamorphosis into benthic juvenile, offshore decapods face many challenges. For the developing individual survivorship will depend heavily on food availability but also on the reserves passed on by the mother. Even though vertical migration behavior can allow the larvae to take advantage of depth varying currents for transport, the effect of general circulation pattern will superimpose local current and influence feeding conditions and affect dispersal and recruitment.

Micropropagation in elite corck oak trees: a tool for suber production improvement?

Dada a extrema importância económica e ambiental que o montado de sobro tem em Portugal, e dado o declínio deste devido a várias razões (e.g. doença, idade das plantas) é premente desenvolver estratégias de preservação de sobreiros elite e optimizar técnicas para a propagação destes genótipos. No primeiro Capítulo expõe-se uma breve introdução sobre o montado actual e as técnicas actuais de regeneração/propagação do sobreiro. Descreve-se ainda as principais técnicas de preservação e avaliação de estabilidade genética referidas na literatura para sobreiro e outras lenhosas. No Capítulo II é apresentado um estudo de melhoramento das condições actuais de maturação de embriões somáticos de sobreiro com vista a aperfeiçoar o processo de conversão em plantas. Neste capítulo é apresentado um protocolo melhorado em relação ao actual que permite um desenvolvimento dos embriões somáticos dum modo semelhante aos embriões zigóticos em termos de substâncias de reserva. O Capítulo III mostra um estudo efectuado com o objectivo principal de avaliar estabilidade genética durante todo o processo de embriogénese somática. Neste capítulo são apresentados resultados duma análise feita por RAPD em fases distintas da embriogénese somática de sobreiro. Neste estudo mostra-se que não existem diferenças significativas entre plantas de campo, embriões somáticos e plantas regeneradas. No Capítulo VI, pretende-se complementar o estudo anterior. Neste Capítulo descreve-se a dinâmica do ciclo celular durante as primeiras fases de embriogénese somática na presença de reguladores de crescimento. Este trabalho permitiu concluir a importância dos reguladores de crescimento na indução e perceber o peso do factor genótipo durante o processo. Considerando os resultados anteriores, a necessidade de um processo eficiente de preservação de genótipos elite torna-se fundamental. No Capítulo V descreve-se um protocolo de criopreservação eficiente sem recursos a substâncias tóxicas. Nesta secção é ainda feita uma análise de variabilidade genética após criopreservação através de FCM, AFLP e SSR. Todos os resultados obtidos anteriormente são postos a prova no Capítulo VI onde se faz uma monitorização extensiva de 10 genótipos elite, tendo em conta a sua capacidade de produção de cortiça, através do processo de embriogénese somática. Durante esta secção são utilizados os protocolos desenvolvidos anteriormente e avaliados na sua eficiência. Neste capítulo é descrita a integração de vários segmentos deste estudo num só protocolo eficiente de regeneração e preservação de sobreiros através de embriogénese somática. Finalmente, no Capítulo VI são apresentadas as conclusões da presente Tese de Doutoramento, com especial incidência para linhas de investigação futuras a serem tomadas. Discute-se a importância deste novo protocolo na optimização da produção da cortiça e traçam-se possíveis aplicações alternativas.

Toxicogenomics of natural and anthropogenic effectors in eleutheroembryos

The introduction of chemicals into the environment by human activities may represent a serious risk to environmental and human health. Environmental risk assessment requires the use of efficient and sensitive tools to determine the impact of contaminants on the ecosystems. The use of zebrafish for the toxicity assessment of pharmaceuticals, drugs, and pollutants, is becoming well accepted due to zebrafish unique advantages for the screening of compounds for hazard identification. The aim of the present work is to apply toxicogenomic approaches to identify novel biomarkers and uncovered potential modes of action of classic and emergent contaminants able to disrupt endocrine systems, such as the Retinoic Acid Receptor, Retinoid X Receptor and the Aryl Hydrocarbon Receptor. This study relies on different nuclear and cytosolic protein receptors and other conditional (ligand- or stress- activated) transcriptional factors that are intimately involved in the regulation of defensome genes and in mechanisms of chemical toxicity. The transcriptomic effects of organic compounds, endogenous compounds, and nanoparticles were analysed during the early stages of zebrafish development. Studying the gene expression profiles of exposed and unexposed organisms to pollutants using microarrays allowed the identification of specific gene markers and to establish a "genetic code" for the tested compounds. Changes in gene expression were observed at toxicant concentrations that did not cause morphological effects. Even at low toxicant concentrations, the observed changes in transcript levels were robust for some target genes. Microarray responses of selected genes were further complemented by the real time quantitative polymerase chain reaction (qRT-PCR) methodology. The combination of bio-informatic, toxicological analyses of differential gene expression profiles, and biochemical and phenotypic responses across the treatments allowed the identification of uncovered potential mechanisms of action. In addition, this work provides an integrated set of tools that can be used to aid management-decision making by improving the predictive capability to measure environmental stress of contaminants in freshwater ecosystems. This study also illustrates the potential of zebrafish embryos for the systematic, large-scale analysis of chemical effects on developing vertebrates.

Characterization of proteotoxic stress in zebrafish

A fidelidade da síntese proteica é fundamental para a estabilidade do proteoma e para a homeostasia celular. Em condições fisiológicas normais as células têm uma taxa de erro basal associada e esta muitas vezes aumenta com o envelhecimento e doença. Problemas na síntese das proteínas estão associados a várias doenças humanas e aos processos de envelhecimento. De facto, a incorporação de erros nas proteínas devido a tRNAs carregados pelas aminoacil-tRNA sintetases com o amino ácido errado causa doenças neurodegenerativas em humanos e ratos. Ainda não é claro como é que estas doenças se desenvolvem e se são uma consequência directa da disrupção do proteoma ou se são o resultado da toxicidade produzida pela acúmulação de proteínas mal traduzidas ao nível do ribossoma. Para elucidar como é que as células eucarióticas lidam com proteínas aberrantes e agregados proteicos (stress proteotóxico) desenvolvemos uma estratégia para destabilizar o proteoma. Para isso estabelecemos um sistema de erros de tradução em embriões de peixe zebra que assenta em tRNAs mutantes capazes de incorporar erradamente serina nas proteínas. As proteínas produzidas neste sistema despoletam as vias de resposta ao stress, nomeadamente a via da ubiquitina-proteassoma (UPP – “ubiquitin protesome pathway”) e a via do retículo endoplasmático (UPR – “unfolded protein response”). O stress proteotóxico gerado pelos erros de tradução altera a expressão génica e perfis de expressão de miRNAs, o desenvolvimento embrionário e viabilidade, aumenta a produção de espécies reactivas de oxigénio (ROS), leva ainda à acumulação de agregados proteicos e à disfunção mitocondrial. As malformações embrionárias e fenótipos de viabilidade que observámos foram revertidos por antioxidantes, o que sugere que os ROS desempenham papéis importantes nos fenótipos degenerativos celulares induzidos pela produção de proteínas aberrantes e agregação proteica. Estabelecemos ainda uma linha de peixe zebra transgénica para o estudo do stress proteotóxico. Este trabalho mostra que a destabilização do proteoma em embriões de peixe zebra com tRNAs mutantes é uma boa metodologia para estudar a biologia do stress proteotóxico visto que permite a agregação controlada do proteoma, mimetizando os processos de agregação de proteínas que ocorrem naturalmente durante o envelhecimento e em doenças conformacionais humanas.

Effects of environmental factors on the toxicity of pesticides to zebrafish embryos

During the last century mean global temperatures have been increasing. According to the predictions, the temperature change is expected to exceed 1.5ºC in this century and the warming is likely to continue. Freshwater ecosystems are among the most sensitive mainly due to changes in the hydrologic cycle and consequently changes in several physico-chemical parameters (e.g. pH, dissolved oxygen). Alterations in environmental parameters of freshwater systems are likely to affect distribution, morphology, physiology and richness of a wide range of species leading to important changes in ecosystem biodiversity and function. Moreover, they can also work as co-stressors in environments where organisms have already to cope with chemical contamination (such as pesticides), increasing the environmental risk due to potential interactions. Therefore, the objective of this work was to evaluate the effects of climate change related environmental parameters on the toxicity of pesticides to zebrafish embryos. The following environmental factors were studied: pH (3.0-12.0), dissolved oxygen level (0-8 mg/L) and UV radiation (0-500 mW/m2). The pesticides studied were the carbamate insecticide carbaryl and the benzimidazole fungicide carbendazim. Stressors were firstly tested separately in order to derive concentration- or intensity-response curves to further study the effects of binary combinations (environmental factors x pesticides) by applying mixture models. Characterization of zebrafish embryos response to environmental stress revealed that pH effects were fully established after 24 h of exposure and survival was only affected at pH values below 5 and above 10. Low oxygen levels also affected embryos development at concentrations below 4 mg/L (delay, heart rate decrease and edema), and at concentrations below 0.5 mg/L the survival was drastically reduced. Continuous exposure to UV radiation showed a strong time-dependent impact on embryos survival leading to 100% of mortality after 72 hours of exposure. The toxicity of pesticides carbaryl and carbendazim was characterized at several levels of biological organization including developmental, biochemical and behavioural allowing a mechanistic understanding of the effects and highlighting the usefulness of behavioural responses (locomotion) as a sensitive endpoint in ecotoxicology. Once the individual concentration response relationship of each stressor was established, a combined toxicity study was conducted to evaluate the effects of pH on the toxicity of carbaryl. We have shown that pH can modify the toxicity of the pesticide carbaryl. The conceptual model concentration addition allowed a precise prediction of the toxicity of the jointeffects of acid pH and carbaryl. Nevertheless, for alkaline condition both concepts failed in predicting the effects. Deviations to the model were however easy to explain as high pH values favour the hydrolysis of carbaryl with the consequent formation of the more toxic degradation product 1- naphtol. Although in the present study such explanatory process was easy to establish, for many other combinations the “interactive” nature is not so evident. In the context of the climate change few scenarios predict such increase in the pH of aquatic systems, however this was a first approach focused in the lethal effects only. In a second tier assessment effects at sublethal level would be sought and it is expectable that more subtle pH changes (more realistic in terms of climate changes scenarios) may have an effect at physiological and biochemical levels with possible long term consequences for the population fitness.

Cannabinoids impact on pregnancy: effects in trophoblast cells

Cannabinoids (CBs) can be classified as: phytocannabinoids, the constituents of the Cannabis sativa plant; synthetic cannabinoids lab-synthesized and the endocannabinoids that are endogenous lipid mediators. Cannabinoid compounds activate cannabinoid receptors – CB1 and CB2. The most prevalent psychoactive phytocannabinoid is Δ9tetrahydrocannabinol (THC), but more than 60 different CBs were already identified in the plant. The best characterized endocannabinoids (eCBs) are anandamide (AEA) and 2arachidonoylglycerol (2-AG), that are involved in several physiological processes including synaptic plasticity, pain modulation, energy homeostasis and reproduction. On the other hand, some synthetic cannabinoids that were initially designed for medical research, are now used as drugs of abuse. During the period of placental development, highly dynamic processes of remodeling occur, involving proliferation, apoptosis, differentiation and invasion of trophoblasts. It is known that a tight control of eCBs levels is required for normal pregnancy progression and that eCBs are involved in trophoblast cells turnover. Therefore, by sharing activation of the same receptors, exposure to exocannabinoids either by recreational or medicinal use may lead to alterations in the eCBs levels and in the endocannabinoid system homeostasis In this work, it was studied the impact of CBs in BeWo trophoblastic cells and in primary cultures of human cytotrophoblasts. Cells were treated for 24 hours with different concentrations of THC, the synthetic cannabinoid WIN‐55,212 (WIN) and 2-AG. Treatment with THC did not affect BeWo cells viability while WIN and 2-AG caused a dose-dependent viability loss. Morphological studies together with biochemical markers indicate that 2-AG is able to induce apoptosis in cytotrophoblasts. On the other hand, morphological studies after acridine orange staining suggest that autophagy may take part in WIN-induced loss of cell viability. All cannabinoids caused a decrease in mitochondrial membrane potential (Δψm) but only 2-AG led to ROS/RNS generation, though no changes in glutathione levels were observed. In addition, ER-stress may be involved in the 2-AG induced-oxidative stress, as preliminary results point to an increase in CCAAT-enhancer-binding protein homologous protein (CHOP) expression. Besides the decrease in cell viability, alterations in cell cycle progression were observed. WIN treatment induced a cell cycle arrest in G0/G1 phase, whereas 2-AG induced a cell cycle arrest in G2/M phase. Here it is reinforced the relevance of cannabinoid signaling in fundamental processes of cell proliferation and cell death in trophoblast cells. Since cannabis-based drugs are the most consumed illicit drugs worldwide and some of the most consumed recreational drugs by pregnant women, this study may contribute to the understanding of the impact of such substances in human reproduction.