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- Intestinal response to ocean acidification in the European sea bass (Dicentrarchus labrax)Publication . Alves, Alexandra; Gregorio, Silvia F.; Ruiz-Jarabo, Ignacio; Fuentes, Juanthe intestine of marine fishes contributes to the ocean carbon cycle producing carbonate aggregates as part of the osmoregulatory process. Therefore, this study aimed to evaluate physiological adjustments of European sea bass (Dicentrarchus labrax) intestine to a higher pCO(2) environment likely in the near future (similar to 1700 mu atm). At the whole-body level, hypercapnia for 5 weeks resulted in fish having a significantly diminished specific growth rate, condition factor and hepatosomatic index. An increase in plasma osmolality and HCO3- concentration was detected, paralleled by decreased metabolites concentrations. In the intestine, high seawater pCO(2) was without effect on ouabain-sensitive ATPase activities, while Bafilomycin A1-sensitive ATPase activity significantly decreased in the anterior intestine. Anterior and mid intestine were mounted in Ussing chambers in order to measure bioelectrical parameters and bicarbonate secretion by pH-Stat ex-vivo. Hypercapnia induced a 2.3 and 2.8-fold increase in bicarbonate secretion rates in the anterior and mid intestine, respectively. In the intestinal fluid, HCO3- concentration increased 2.2-fold, and carbonate precipitates showed a 4.4-fold increase in response to hypercapnia, paralleled by a > 3-fold increase of drinking and a > 2-fold increase of intestinal volume at any given time. At the molecular level, hypercapnia elicited higher intestinal mRNA expression levels for atp6v1b (V-ATPase B subunit), slc4a4, slc26a3, and slc26a6, both in the anterior and mid intestine. As a whole, our results show that the intestine of sea bass responds to high seawater pCO(2), a response that comes at a cost at the whole-body level with an impact in the fish specific growth rate, condition factor, and hepatosomatic index.
- Modulação da transmissão colinérgica no órgão eléctrico de Torpedo marmorata (Risso, 1810)Publication . Alves, Alexandra Carvalho; Cordeiro, João Miguel da Silva; Canario, Adelino V. M.Este estudo incidiu nos mecanismos envolvidos na modulação da transmissão colinérgica por receptores nicotínicos (nAChR) pré-sinápticos. Neste trabalho, utilizaram-se os electrócitos do órgão eléctrico de Torpedo marmorata, como detectores de libertação de ACh em tempo real através da medição de potenciais de electroplaca (EPP) numa pilha de electrócitos sobrepostos (prisma). Estes potenciais resultam da libertação síncrona de ACh a partir de uma miríade de sinapses e possibilitam estudos sobre a neuromodulação nicotínica com uma resolução de milisegundos. Para tal, avaliou-se o efeito da aplicação do agonista dos nAChR, iodeto de 1,1- dimetil-4-fenilpiperazínio (DMPP), durante 5 s sobre os potenciais eléctricos dos prismas. A presença de DMPP aumentou a amplitude (~ 20%) e a área (~ 37%) dos EPP, sendo este aumento dependente da concentração de agonista (n=10). O efeito facilitatório do DMPP (0,1-10 μM) na amplitude e na área dos prismas aumentou com a redução da concentração extracelular de Ca2+ de 3,4 mM (concentração fisiológica normal) para 1,7, 1,275 e 0,85 mM. Os resultados obtidos quando se avaliou a razão área/pico e latência demonstraram que os parâmetros relacionados com o tempo do sinal foram menos sensíveis à diminuição do Ca2+ extracelular. O efeito facilitatório da ACh libertada endogenamente foi prevenido na presença dos antagonistas nicotínicos, dihidro- beta-eritroidina (DH-β-E) e hexametónio (HE). Estes resultados mostram que a acetilcolina (ACh) aumenta a sua própria libertação através da activação de nAChR. Este mecanismo pode funcionar como um amplificador pré-sináptico, aumentando a margem de segurança da transmissão sináptica, semelhante ao que foi observado na junção neuromuscular do rato (Faria et al., 2003). Para avaliar se as respostas eléctricas dos prismas ao DMPP teriam origem pré ou pós-sináptica, bloqueou-se a condução do impulso nervoso com TTX (1 μM) e inibiu-se a actividade da acetilcolinesterase (AChE) com neostigmina (20 μM). Nestas circunstâncias, a aplicação de ACh (100 μM) produziu potenciais pós-sinápticos superiores a 100 mV durante alguns segundos, ao contrário do que se observou após a aplicação de DMPP (100 μM) (n=4). Os resultados mostram claramente que o DMPP actua pré-sinapticamente facilitando a libertação de ACh, e que a sua actividade póssináptica é negligenciável. Para confirmar esta hipótese, realizaram-se ensaios com terminais nervosos isolados (sinaptossomas) do órgão eléctrico de Torpedo, onde foi possível medir directamente a libertação de ACh através de um método de quimioluminescência. Nestas circunstâncias, o DMPP aumentou a libertação de ACh (~ 30%) induzida através da despolarização dos sinaptossomas com veratridina (100 μM) ou KCl (40 mM), confirmando a natureza pré-sináptica da acção do DMPP. À semelhança do que se observou nas experiências electrofisiológicas com os prismas de Torpedo, o efeito facilitatório do DMPP nos sinaptossomas foi prevenido pela DH-β-E (3 μM), um antagonista dos nAChR contendo sub-unidades α3/4β2 dos nAChR. A relação espacial entre os nAChR e as zonas activas de libertação de ACh foi avaliada electrofisiológicamente usando os quelantes de Ca2+ BAPTA-AM e EGTAAM. Esta metodologia, demonstrou a influência dos microdomínios de Ca2+ na modulação da libertação de ACh pelos nAChR pré-sinápticos. Os sensores de Ca2+ (da libertação de ACh) aparentam não estar co-localizados com os nAChR. Este trabalho constitui a primeira tentativa, de que se tem conhecimento, para avaliar o papel dos autoreceptores nicotínicos no órgão eléctrico do T. marmorata. Serão necessárias mais experiências para caracterizar melhor o subtipo de nAChR envolvido na facilitação de ACh nesta preparação.
- Molecular and functional regionalization of bicarbonate secretion cascade in the intestine of the European sea bass (Dicentrarchus labrax)Publication . Alves, Alexandra; Gregorio, Silvia F.; Catão-Egger, Renata; Fuentes, JuanIn marine fish the intestinal HCO3-(-) secretion is the key mechanism to enable luminal aggregate formation and water absorption. Using the sea bass (Dicentrarchus labrax), the present study aimed at establishing the functional and molecular organization of different sections of the intestine concerning bicarbonate secretion and Cl- movements. The proximal intestinal regions presented similar HCO3-- secretion rates, while differences were detected in the molecular expression of the transporters involved and on regional HCO3- concentrations. The anterior region presented significantly higher Na /K+-ATPase activity, Cl transepithelial transport and basolateral slc4a4, apical slc26a6 and slc26a3 expression levels. In the mid intestine, the total HCO3- content was significantly increased in the fluid as in the carbonate aggregates. In the rectum no HCO3- secretion was observed and was characterized by the diminished HCO3- total content, residual molecular expression of slc4a4, slc26a6 and slc26a3, higher H+-ATPase activity and expression, suggesting the existence of a different bicarbonate handling mechanism. The possible regulation of HCO3- secretion by extracellular HCO3- and increased intracellular cAMP levels were also investigated. cAMP did not affect HCO3- secretion, although Cl- secretion was enhanced by cftr. HCO3- secretion rise due to the HCO3- basolateral increment showed that at resting levels slc4a4 was not a limiting step for secretion. The transcellular/intracellular dependence of apical HCO3- secretion differed between the proximal regions. In conclusion, intestinal HCO3- secretion has a functional region-dependent organization that was not reflected by the anterior-posterior regionalization on HCO3- secretion and expression profiles of chloride/water absorption related genes.
- Ocean acidification compromises energy management in Sparus aurata (Pisces: Teleostei)Publication . Ruiz-Jarabo, I.; Gregorio, SF; Alves, Alexandra; Mancera, J. M.; Fuentes, JuanThe effects of ocean acidification mediated by an increase in water pCO(2) levels on marine organisms are currently under debate. Elevated CO2 concentrations in the seawater induce several physiological responses in teleost fish, including acid-base imbalances and osmoregulatory changes. However, the consequences of CO2 levels enhancement on energy metabolism are mostly unknown. Here we show that 5 weeks of exposure to hypercapnia (950 and 1800 mu atm CO2) altered intermediary metabolism of gilthead seabream (Sparus aurata) compared to fish acclimated to current ocean values (440 mu atm CO2). We found that seabream compromises its physiological acid-base balance with increasing water CO2 levels and the subsequent acidification. Intestinal regions (anterior, mid, and rectum) engaged in maintaining this balance are thus altered, as seen for Na+/K+ ATPase and the vacuolar-type H+-ATPase activities. Moreover, liver and muscle counteracted these effects by increasing catabolic routes e.g., glycogenolysis, glycolysis, amino acid turnover, and lipid catabolism, and plasma energy metabolites were altered. Our results demonstrate how a relatively short period of 5 weeks of water hypercapnia is likely to disrupt the acid-base balance, osmoregulatory capacity and intermediary metabolism in S. aurata. However, long-term studies are necessary to fully understand the consequences of ocean acidification on growth and other energy-demanding activities, such as reproduction.
- Refining tools for studying cuttlefish ( Sepia officinalis ) reproduction in captivity: in vivo sexual determination, tagging and DNA collectionPublication . Sykes, António V.; Alves, Alexandra; Capaz, Juan Carlos; Madeira, Céline; Couto, Ana T.; Gonçalves, Rui A.; Frias, Paulo A.; Leal, Irene; Andrade, José PedroTo overcome the bottleneck of reproduction in cuttlefish, Sepia officinalis, aquaculture development, there is need for new tools or refinement of methods. While attaining low welfare impact on individuals, procedures to allow for in vivo individual sexual and maturity determination, tagging and collection of samples for high quality DNA are needed. The present study evaluated the use of an endoscope, visual implant elastomer and swabbing for each, respectively, in a set of 3 experiments that registered its effects on growth, mortality and reproduction. Results showed that all the methodologies tested herein were of easy application, did not promote injuries nor poor growth, abnormal reproduction or increased mortality. This indicates that cuttlefish would not be experiencing pain, suffering, distress and lasting harm (PSDLH), resulting from the application of these procedures and that these are therefore recommended as new standards for reproduction studies in the species.