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- Asparagopsis armata exudate cocktail: the quest for the mechanisms of toxic action of an invasive seaweed on marine invertebratesPublication . Silva, Carla; Simões, Tiago; Félix, Rafael; A.M.V.M., Soares; Barata, Carlos; Novais, Sara C.; Lemos, Marco F.L.The seaweed Asparagopsis armata exhibits a strong invasive behavior, producing halogenated compounds with effective biological effects. This study addresses the biochemical responses to sublethal concentrations of A. armata exudate on the marine snail Gibbula umbilicalis whole body and the shrimp Palaemon elegans eyes and hepatopancreas. Antioxidant defenses superoxide dismutase (SOD) and glutathione-S-transferase (GST), oxidative damage endpoints lipid peroxidation (LPO) and DNA damage, the neuronal parameter acetylcholinesterase (AChE), and the fatty acid profile were evaluated. Results revealed different metabolic responses in both species. Despite previous studies indicating that the exudate affected G. umbilicalis’ survival and behavior, this does not seem to result from oxidative stress or neurotoxicity. For P. elegans, the inhibition of AChE and the decrease of antioxidant capacity is concomitant with the increase of LPO, suggesting neurotoxicity and oxidative stress as contributor mechanisms of toxicity for this species. Fatty acid profile changes were more pronounced for P. elegans with a general increase in polyunsaturated fatty acids (PUFAs) with the exudate exposure, which commonly means a defense mechanism protecting from membrane disruption. Nonetheless, the omega-3 PUFAs arachidonic acid (ARA) and docosapentaenoic acid (DPA) increased in both invertebrates, indicating a common regulation mechanism of inflammation and immunity responses
- Effect of three diets on the growth and fatty acid profile of the common ragworm Hediste diversicolor (O.F. Müller, 1776)Publication . Santos, António; Granada, Luana; Baptista, Teresa; Anjos, Catarina; Simões, Tiago; Tecelão, Carla; Fidalgo e Costa, Pedro; Costa, José Lino; Pombo, AnaThe polychaete Hediste diversicolor has a high physiological tolerance to extreme environmental factors, being easily farmed and reproduced in different types of conditions. Both in the field and under laboratory conditions, this worm can feed on different types of food. In order to highlight the potential of H. diversicolor for aquaculture, specific growth rate (SGR), daily growth rate (DGR), survival rate and fatty acid profile of juvenile worms, fed with three different diets, were assessed. The experiments were conducted using juvenile polychaete from a controlled reproduction with wild adults. H. diversicolor individuals were fed with two commercial diets, seabream dry feed (Aquagold) and semi-wet pellets for reared sole (Moist Sole), and with a non-processed diet consisting on mackerel's fillets (Trachurus trachurus). Juveniles fed with Aquagold had the highest final individual weight (0.89 ± 0.10 g). The SGR was higher in H. diversicolor fed with Aquagold and Moist Sole, (6.49 ± 0.30% d−1 and 6.54 ± 0.06% d−1 , respectively. The highest DGR was observed for juveniles fed with Aquagold (0.146 ± 0.02 g d−1 ). The survival rate of ragworms under different treatments ranged from 96 to 100%. Regarding the protein content, the Moist Sole diet provided the highest percentage of protein in the reared worms (8.87%). Results showed that the total fat content of the diets was reflected in the fat content of the reared worms. The Moist Sole diet treatment had the highest fat content (2.25%) and individuals fed with seabream dry feed showed similar results (2.18%), while the lowest percentage was observed for the mackerel diet (0.85%). According to the fatty acid profile, the major fatty acids found in the juveniles fed with the three different diets were palmitic (C 16:0), with a higher value in the individuals fed with mackerel's fillets. Oleic (C 18:1 n9), eicosapentaenoic (C 20:5 n3), docosahexaenoic (C 22:6 n3) and stearic (C 18:0) acids presented high values in H. diversicolor fed with all the experimental diets. Statement of Relevance: The common ragworm Hediste diversicolor is a potential high quality fatty acids source for reared fish and shrimp. Previous studies suggested that diet could be a relevant factor affecting the fatty acid composition of this polychaete (Luis and Passos, 1995). This study aimed to assess the effect of different diets on growth and survival of common ragworms juveniles (H. diversicolor), as well as the fatty acid profile and protein content in their tissues, aiming to find an appropriate diet to be used in commercial aquaculture.
- From laboratory to the field: Validating molecular markers of effect in Folsomia candida exposed to a fungicide-based formulationPublication . Simões, Tiago; Novais, Sara C.; Natal-da-Luz, Tiago; Renaud, Mathieu; Leston, Sara; Ramos, Fernando; Römbke, Jörg; Dick, Roelofs; Van Straalen, Nico M.; Sousa, José P.; Lemos, Marco F. L.Under controlled laboratory conditions, toxicity data tend to be less variable than in more realistic in-field studies and responses may thus differ from those in the natural environment, creating uncertainty. The validation of data under environmental conditions is therefore a major asset in environmental risk assessment of chemicals. The present study aimed to validate the mode of action of a commercial fungicide formulation in the soil invertebrate F. candida, under more realistic exposure scenarios (in-field bioassay), by targeting specific molecular biomarkers retrieved from laboratory experiments. Organisms were exposed in soil cores under minimally controlled field conditions for 4 days to a chlorothalonil fungicide dosage causing 75% reduction of reproduction in a previous laboratory experiment (127 mg a.i. kg−1 ) and half this concentration (60 mg a.i. kg−1 ). After exposure, organisms were retrieved and RNA was extracted from each pool of organisms. According to previous laboratorial omics results with the same formulation, ten genes were selected for gene expression analysis by qRT-PCR, corresponding to key genes of affected biological pathways including glutathione metabolism, oxidation-reduction, body morphogenesis, and reproduction. Six of these genes presented a dose-response trend with higher up- or down-regulation with increasing pesticide concentrations. Highly significant correlations between their expression patterns in laboratory and in-field experiments were observed. This work shows that effects of toxicants can be clearly demonstrated in more realistic conditions using validated biomarkers. Our work outlines a set of genes that can be used to assess the early effects of pesticides in a realistic agricultural scenario.
- Using biomarkers to address the impacts of pollution on limpets ( Patella depressa ) and their mechanisms to cope with stressPublication . Silva, Cátia S. E.; Novais, Sara C.; Simões, Tiago; Caramalho, Márcia; Gravato, Carlos; Rodrigues, Maria J.; Maranhão, Paulo; Lemos, Marco F. L.Contaminant discharges, derived from human activities, are major concerns as they exert a continuous ecological pressure on aquatic systems, causing in fact cascading community effects. Although with associated ecological and fitness costs, physiological and biochemical defense mechanisms may restore the organism’s internal balance. Several studies have pointed biomarkers as tools to assess contamination levels, and also to understand potential mechanisms to cope with stress. In the present study, the fitness costs of limpets Patella depressa located in two different contaminated sites and when transplanted into a different common environment – with no known historical contamination – were assessed through the measurement of oxidative stress, energy metabolism responses, and fatty acid profile changes. Integrated biomarker response index (IBR) revealed differences in responses between organisms of the different origin sites, with greater antioxidant and detoxification activities in the site with the higher contamination life-history (higher IBR index). Moreover, different abilities of this species to deal with the new environmental condition were also observed. After the transplant, and despite the differences in the initial profiles, response patterns became similar between both populations (similar IBR index), with organisms from the less contaminated site suffering a higher impact with a demarked increase in their detoxification and antioxidant defenses, as well as higher changes on fatty acid abundance/composition. Through an integrated biomarker profile analysis in a transplant context, this work provides a distinct insight on the mechanisms of response and tolerance to environmental stress, and fitness costs of this potential sentinel marine species in the context of environmental contamination changes.
- Using time-lapse omics correlations to integrate toxicological pathways of a formulated fungicide in a soil invertebratePublication . Simões, Tiago; Novais, Sara C.; Natal-da-Luz, Tiago; Devreese, Bart; de Boer, Tjalf; Roelofs, Dick; Sousa, José P.; Van Straalen, Nico M.; Lemos, Marco F. L.The use of an integrative molecular approach can actively improve the evaluation of environmental health status and impact of chemicals, providing the knowledge to develop sentinel tools that can be integrated in risk assessment studies, since gene and protein expressions represent the first response barriers to anthropogenic stress. This work aimed to determine the mechanisms of toxic action of a widely applied fungicide formulation (chlorothalonil), following a time series approach and using a soil model arthropod, Folsomia candida. To link effects at different levels of biological organization, data were collected on reproduction, gene expression and protein levels, in a time series during exposure to a natural soil. Results showed a mechanistic mode of action for chlorothalonil, affecting pathways of detoxification and excretion, immune response, cellular respiration, protein metabolism and oxidative stress defense, causing irregular cell signaling (JNK and NOD ½ pathways), DNA damage and abnormal cell proliferation, leading to impairment in developmental features such as molting cycle and reproduction. The omics datasets presented highly significant positive correlations between the gene expression levels at a certain time-point and the corresponding protein products 2e3 days later. The integrated omics in this study has provided useful insights into pesticide mechanisms of toxicity, evidencing the relevance of such analyses in toxicological studies, and highlighting the importance of considering a time-series when integrating these datasets.
- Fate and effects of two pesticide formulations in the invertebrate Folsomia candida using a natural agricultural soilPublication . Simões, Tiago; Novais, Sara C.; Natal-da-Luz, Tiago; Leston, Sara; Rosa, João; Ramos, Fernando; Pouca, Ana Sofia Vila; Freitas, Andreia; Barbosa, Jorge; Roelofs, Dick; Sousa, José P.; Van Straalen, Nico M.; Lemos, Marco F. L.Degradation rates of two widely used pesticides were assessed, and acute and chronic effects on a standard invertebrate species investigated. An herbicide (Montana®) and fungicide (Bravo500®) formulations were investigated and results were compared to the isolated active substances of each formulation (glyphosate and chlorothalonil, respectively). Tests were performed using the invertebrate Folsomia candida as test species and an agricultural natural soil. Degradation rate tests were determined under aerobic conditions at 20 ± 2 °C, using an ecologically relevant concentration of 5 mg (a.i.) kg−1 of soil for both chemicals. Results demonstrated degradation half-lives (DT50) of 2.2 days for Montana® and 2.8 days when pure glyphosate was tested. Values of 1.1 and 2.9 days were registered for Bravo500® and its active substance chlorothalonil, respectively. There were no effects on survival for the tested concentrations of both forms of the herbicide (up to 17.3 mg kg−1). However, reproduction was affected, but only by the herbicide formulation, with an estimated EC50 value of 4.63 mg (a.i.) kg−1. Effects were most unlikely related to glyphosate. For chlorothalonil, both tested forms affected survival and reproduction. The estimated LC50 values were 117 mg (a.i.) kg−1 and 73.5 mg (a.i.) kg−1, and the EC50 41.3 mg (a.i.) kg−1 and 14.9 mg kg−1 for the formulation and the active ingredient, respectively. The effects of the active ingredient were significantly stronger, indicating the major influence of the active substance in the effects caused also by the formulation. Overall results demonstrate the importance of evaluating the effects of the formulated chemicals, as they are applied in the field, and not only their isolated active ingredients.
- Allometric relationships to assess ontogenetic adaptative changes in three NE Atlantic commercial sea cucumbers (Echinodermata, Holothuroidea)Publication . Silva, F. Azevedo e; Brito, A. C.; Simões, T.; Pombo, A.; Marques, T. A.; Rocha, C.; Sousa, J.; Venâncio, E.; Félix, P. M.Holothuria arguinensis, Holothuria mammata and Holothuria forskali are three common sea cucumber species found in the NE Atlantic, traded in international markets and susceptible to capture. Allometric relationships reveal if the scaling relationships between biometric characters are proportional with growth, being a useful tool to understand species growth strategies. Allometric relationships of the three species were estimated and compared between them and with populations from different regions. These allometric relationships revealed that the three species have negative allometric growth. However, they have different growth strategies between them and reveal different regional intra-specific growth strategies when compared to other populations, suggesting ontogenetic adaptation as a consequence of external factors.
- Sex-steroids and hypolipidemic chemicals impacts on brown trout lipid and peroxisome signaling — Molecular, biochemical and morphological insightsPublication . Madureira, Tânia Vieira; Malhão, Fernanda; Simões, Tiago; Pinheiro, Ivone; Lopes, Célia; Gonçalves, José F.; Urbatzka, Ralph; Castro, L. Filipe C.; Lemos, Marco F.L.; Rocha, EduardoLipid metabolism involves complex pathways, which are regulated in a similar way across vertebrates. Hormonal and hypolipidemic deregulations cause lipid imbalance from fish to humans, but the underlying mechanisms are far from understood. This study explores the potential of using juvenile brown trout to evaluate the in vivo interferences caused by estrogenic (17α-ethinylestradiol - EE2), androgenic (testosterone - T), and hypolipidemic (clofibrate - CLF) compounds in lipidic and/or peroxisomal pathways. Studied endpoints were from blood/plasma biochemistry, plasma fatty acid profile, ultrastructure of hepatocytes and abundance of their peroxisomes to mRNA expression in the liver. Both T and CLF caused minimal effects when compared to EE2. Estrogenized fish had significantly higher hepatosomatic indexes, increased triglycerides and very-low density lipoproteins (VLDL) in plasma, compared with solvent control. Morphologically, EE2 fish showed increased lipid droplets in hepatocytes, and EE2 and T reduced volume density of peroxisomes in relation to the hepatic parenchyma. Polyunsaturated fatty acids (PUFA) in plasma, namely n-3 PUFA, increased with EE2. EE2 animals had increased mRNA levels of vitellogenin A (VtgA), estrogen receptor alpha (ERα), peroxisome proliferator-activated receptor alpha (PPARα), PPARαBa and acyl-CoA long chain synthetase 1 (Acsl1), while ERβ-1, acyl-CoA oxidase 1-3I (Acox1-3I), Acox3, PPARγ, catalase (Cat), urate oxidase (Uox), fatty acid binding protein 1 (Fabp1) and apolipoprotein AI (ApoAI) were down-regulated. In summary, in vivo EE2 exposure altered lipid metabolism and peroxisome dynamics in brown trout, namely by changing the mRNA levels of several genes. Our model can be used to study possible organism-level impacts, viz. in gonadogenesis.
- Síntese enzimática de lípidos estruturados sucedâneos de gordura de leite humano, enriquecidos em ácidos gordos polinsaturadosPublication . Simões, TiagoNa gordura de leite humano (HMF- “Human Milk Fat”), o ácido palmítico encontrase esterificado principalmente na posição interna (sn-2) dos triacilgliceróis, enquanto os ácidos gordos insaturados ocupam as posições externas (sn-1,3). Os ácidos gordos polinsaturados (PUFA- “Polyunsaturated Fatty Acids”) de cadeia longa, como o ácido araquidónico (ARA), ácido eicosapentaenóico (EPA) e ácido docosahexaenóico (DHA) são considerados essenciais ao desenvolvimento do recémnascido. No presente trabalho pretendeu produzir-se sucedâneos de gordura de leite humano (HMFS- “Human Milk Fat Substitutes”) por reação de acidólise enzimática entre a banha e um concentrado de óleo de peixe rico em PUFA, em meio livre de solvente. Testaram-se lipases comerciais imobilizadas de Rhizomucor miehei (Lipozyme RM IMTM), de Thermomyces lanuginosa (Lipozyme TL IMTM), de Candida antárctica (Novozym 435TM), e a lipase de Pseudomonas fluorescens (Amano AK TM) não imobilizada. Foram também testados biocatalisadores não comerciais, entre os quais a lipase/ aciltransferase de Candida parapsilosis imobilizada em Accurel® MP 1000, a lipase de Carica papaya autoimobilizada no látex da papaia e a lipase heteróloga de Rhizopus oryzae (rROL) expressa numa estirpe mutante de Pichia pastoris e imobilizada em Accurel® MP 1000. Após 24h de reacção a 50ºC obtiveram-se incorporações molares de PUFA de 17,48% para a “Novozym 435TM”, de 16,92% para a rROL e de 16,75% para a “Lipozyme RM IM TM”. As restantes incorporações variaram entre 8,01% (C. papaya) e 4,37% (Amano AK). A rROL foi o biocatalisador selecionado para ensaios de modelação e otimização das condições reacionais pela Metodologia das Superfícies de Resposta (RSM- “Response surface Methodology”) por se tratar de um biocatalisador não comercial, de baixo custo e sobre o qual existem poucos estudos publicados. A lipase apresentou melhor atividade de acidólise para uma temperatura de 40ºC e razão molar 1:3 (banha/PUFA). Quer o aumento da temperatura como da razão molar conduziram a menores incorporações de PUFA nos TAG. Estudou-se a estabilidade operacional de rROL em ensaios de reutilização sucessiva. Observou-se uma redução de cerca de 20% na actividade residual do biocatalisador, entre a primeira e a segunda reutilização e entre a terceira e a quarta. A seleção do biocatalisador deve ser feita em função da sua atividade de interesterificação e da estabilidade operacional. rROL é um biocatalisador com elevada potencialidade para a síntese de HMFS.
- Fatty acid profile of the sea snail Gibbula umbilicalis as a biomarker for coastal metal pollutionPublication . Silva, Carla; Simões, Tiago; Novais, Sara C.; Pimparel, Inês; Granada, Luana; Soares, Amadeu M. V. M.; Barata, Carlos; Lemos, Marco F. L.Metals are among the most common environmental pollutants with natural or anthropogenic origin that can be easily transferred through the food chain. Marine gastropods are known to accumulate high concentrations of these metals in their tissues. Gibbula umbilicalis ecological importance and abundant soft tissues, which enables extent biochemical assessments, makes this particular organism a potentially suitable species for marine ecotoxicological studies. Fatty acids are carbon-rich compounds that are ubiquitous in all organisms and easy to metabolize. Their biological specificity, relatively well-studied functions and importance, and the fact that they may alter when stress is induced, make fatty acids prospect biomarkers. This work aimed to assess fatty acid profile changes in the gastropod G. umbilicalis exposed to three metal contaminants. After a 168 h exposure to cadmium, mercury, and nickel, the following lipid related endpoints were measured: total lipid content; lipid peroxidation; and fatty acid profile (FAP). The analysis of the FAP suggested an alteration in the fatty acid metabolism and indicated a link between metals exposure and homeoviscous adaptation and immune response. In particular, five fatty acids (palmitic, eicosatrienoic, arachidonic, eicosapentaenoic, and docosahexaenoic acids), demonstrated to be especially good indicators of G. umbilicalis responses to the array of metals used, having thus the potential to be used as biomarkers for metal contamination in this species. This work represents a first approach for the use of FAP signature as a sensitive and informative parameter and novel tool in environmental risk assessment (ERA) of coastal environments, using G. umbilicalis as model species.