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Martin-Robles A.J.,University of Cádiz | Martin-Robles A.J.,Campus Universitario Of Puerto Real | Whitmore D.,University College London | Pendon C.,University of Cádiz | Munoz-Cueto J.A.,Campus Universitario Of Puerto Real
Chronobiology International | Year: 2013

Studies on the developmental onset of the teleost circadian clock have been carried out in zebrafish and, recently, in rainbow trout and Senegalese sole, where rhythms of clock gene expression entrained by light-dark (LD) cycles have been reported from the first days post fertilization. However, investigations of molecular clock rhythms during crucial developmental phases such as metamorphosis are absent in vertebrates. In this study, we documented the daily expression profile of Per1, Per2, Per3, and Clock during Senegalese sole pre-, early-, middle-, and post-metamorphic stages under LD 14:10 cycles (LD group), as well as under transient exposure to constant light (LL-LD group) or constant dark (DD-LD group) conditions. Our results revealed that robust rhythms of clock genes were maintained along the metamorphic process, although with declining amplitudes and expression levels. All daily profiles were affected by transient constant conditions, in particular Per1, Per3, and Clock amplitudes and Per2 acrophase. Rhythm parameters were progressively restored upon reversion to LD cycles but even after 9 d under cycling conditions, a prolonged effect on clock function was observed, especially in the LL-LD group. These results reflect the differential sensitivity of clock machinery of sole to transitory light cues, being Per1 and Per3 predominantly clock regulated and supporting the role of Per2 as part of the light input pathway. Interestingly, there is no reversal in the phase of clock gene rhythms between pre- and post-metamorphic animals that would be coincident with the switch from diurnal to nocturnal locomotor activity, which occurs in this species just before the beginning of this process. Whether specialized central pacemakers dictate the phase of locomotor activity or this control is exerted outside of the core clock mechanism remains to be elucidated. Our results emphasize the importance of maintaining cycling light-dark conditions in aquaculture practices during ontogeny of Senegalese sole. © Informa Healthcare USA, Inc.


Martin-Robles A.J.,University of Cádiz | Martin-Robles A.J.,Campus Universitario Of Puerto Real | Whitmore D.,University College London | Sanchez-Vazquez F.J.,University of Murcia | And 3 more authors.
Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology | Year: 2012

An extensive network of endogenous oscillators governs vertebrate circadian rhythmicity. At the molecular level, they are composed of a set of clock genes that participate in transcriptional-translational feedback loops to control their own expression and that of downstream output genes. These clocks are synchronized with the environment, although entrainment by external periodic cues remains little explored in fish. In this work, partial cDNA sequences of clock genes representing both positive (Clock) and negative (Period1, Period2) elements of the molecular feedback loops were obtained from the nocturnal flatfish Senegalese sole, a relevant species for aquaculture and chronobiology. All of the above genes exhibited high identities with their respective teleost clock genes, and Per-Arnt-Sim or basic helix-loop-helix binding domains were recognized in their primary structure. They showed a widespread distribution through the animal body and some of them displayed daily mRNA rhythms in central (retina, optic tectum, diencephalon, and cerebellum) and peripheral (liver) tissues. These rhythms were most robust in retina and liver, exhibiting marked Period1 and Clock daily oscillations in transcript levels as revealed by ANOVA and cosinor analysis. Interestingly, expression profiles were inverted in retina and optic tectum compared to liver. Such differences suggest the existence of tissue-dependent zeitgebers for clock gene expression in this species (i. e., light for retina and optic tectum and feeding time for liver). This study provides novel insight into the location of the molecular clocks (central vs. peripheral) and their different phasing and synchronization pathways, which contributes to better understand the teleost circadian systems and its plasticity. © 2012 Springer-Verlag.


Trombini C.,Institute Ciencias Marinas Of Andalucia Csic | Hampel M.,Institute Ciencias Marinas Of Andalucia Csic | Hampel M.,Campus Universitario Of Puerto Real | Blasco J.,Institute Ciencias Marinas Of Andalucia Csic
Chemosphere | Year: 2016

The individual and combined toxicities of acetaminophen, carbamazepine, diclofenac and ibuprofen have been examined in neonate nauplii (<24 h-old) of the harpacticoid copepod Tisbe battagliai. Based on acute toxicity data (LC50) obtained, diclofenac was the most toxic compound with an LC50 value of 9.5 mg·L-1; this is between 5 and 7 times lower than the LC50 value for acetaminophen, carbamazepine and ibuprofen (67.8 mg·L-1, 59 mg·L-1 and 49.7 mg·L-1 respectively). The environmental risk posed by the selected pharmaceuticals was assessed by calculating risk quotients (RQs) based on MEC (the highest exposure concentration of the compound in the medium)/PNEC (predicted no effect concentration) ratios. Results suggest that, at environmental concentrations, none of the compounds is harmful for the aquatic environment (low or no risk). Toxicity data obtained for mixtures were compared with predictions derived from three different models: Concentration Addition (CA), Independent Action (IA) and Combination Index (CI). The classical modeling approaches CA and IA failed to predict the observed mixture toxicity, thus indicating that single compound toxicity data are not sufficient to predict toxicity of drug mixtures on Tisbe species. However, the use of the CI seems to provide better predictions of pharmaceutical toxicity. © 2016 Elsevier Ltd.


Carballeira C.,University of Cádiz | Martin-Diaz L.,University of Cádiz | Martin-Diaz L.,Campus Universitario Of Puerto Real | DelValls T.A.,University of Cádiz
Marine Environmental Research | Year: 2011

Sea urchin embryo-larval development (ELD) and fertilization tests have been widely used in ecotoxicity studies and are included in regulatory frameworks. Biological processes occur naturally within a range of salinity that depends on the species considered. In an attempt to determine the optimum range of salinity, ELD and fertilization bioassays were performed at different salinities (15-40.5‰) with two species of Atlantic sea urchin: Arbacia lixula and Paracentrotus lividus. In the ELD assay, the optimum range of salinity was wider for A. lixula (29-35.5‰) than for P. lividus (29-33‰). In the fertilization assay with P. lividus as a bioindicator species, the highest percentage of fertilization (90%) was obtained at salinities of between 29 and 33‰. More research on A. lixula is required, since the fertilization success was below 60%. The results of the present study demonstrate that salinity may be a confounding factor in interpreting ELD test results. © 2011 Elsevier Ltd.


Servili A.,University of Cádiz | Servili A.,Institute Federatif Of Recherche 140 | Herrera-Perez P.,University of Cádiz | Herrera-Perez P.,Campus Universitario Of Puerto Real | And 3 more authors.
General and Comparative Endocrinology | Year: 2012

The European sea bass expresses three GnRH (Gonadotrophin Releasing Hormone) forms that exert pleiotropic actions via several classes of receptors. The GnRH-1 form is responsible for the endogenous regulation of gonadotrophin release by the pituitary gland but the role of GnRH-2 and GnRH-3 remains unclear in fish. In a previous study performed in sea bass, we have provided evidence of direct links between the GnRH-2 cells and the pineal organ and demonstrated a functional role for GnRH-2 in the modulation of the secretory activity of this photoreceptive organ. In this study, we have investigated the possible relationship between the GnRH-3 system and the retina in the same species. Thus, using a biotinylated dextran-amine tract-tracing method, we reveal the presence of retinopetal cells in the terminal nerve of sea bass, a region that also contains GnRH-3-immunopositive cells. Moreover, GnRH-3-immunoreactive fibers were observed at the boundary between the inner nuclear and the inner plexiform layers, and also within the ganglion cell layer. These results strongly suggest that the GnRH-3 neurons located in the terminal nerve area represent the source of GnRH-3 innervation in the retina of this species. In order to clarify whether the retina is a target for GnRH, the expression pattern of GnRH receptors (dlGnRHR) was also analyzed by RT-PCR and in situ hybridization. RT-PCR revealed the retinal expression of dlGnRHR-II-2b, -1a, -1b and -1c, while in situ hybridization only showed positive signals for the receptors dlGnRHR-II-2b and -1a. Finally, double-immunohistochemistry showed that GnRH-3 projections reaching the sea bass retina end in close proximity to tyrosine hydroxylase (dopaminergic) cells, which also expressed the dlGnRHR-II-2b receptor subtype. Taken together, these results suggest an important role for GnRH-3 in the modulation of dopaminergic cell activities and retinal functions in sea bass. © 2011 Elsevier Inc.


Aguirre-Martinez G.V.,University of Cádiz | Aguirre-Martinez G.V.,Campus Universitario Of Puerto Real | Buratti S.,University of Bologna | Fabbri E.,University of Bologna | And 3 more authors.
Environmental Monitoring and Assessment | Year: 2013

The presence of pharmaceuticals in the environment is now a major concern given their potential adverse effects on organisms, particularly human beings. Because the feeding style and habitat of the crab Carcinus maenas make this species vulnerable to organic contaminants, it has been used previously in ecotoxicological studies. Lysosomal membrane stability (LMS) in crabs is a general indicator of cellular well-being and can be visualized by the neutral red retention (NRR) assay. LMS in crab hemolymph has been evaluated as a cellular biomarker of adverse effects produced by exposure to pharmaceutical compounds. Crabs were exposed in the laboratory to four different pharmaceuticals for 28 days in a semistatic 24-h renewal assay. Filtered seawater was spiked every 2 days with various concentrations (from 0.1 to 50 μg·L-1) of caffeine, ibuprofen, carbamazepine, and novobiocin. Results showed that NRR time, measured at day 28, was significantly reduced (p < 0.05) after exposure to environmental concentrations of each pharmaceutical (caffeine=15 μg·L-1; carbamazepine=1 μg·L-1; ibuprofen=5 μg·L-1; and novobiocin=0.1 μg·L-1) when compared with control organisms. The predicted "no environmental effect" concentration/measured environmental concentration results showed that the selected pharmaceuticals are toxic at environmental concentrations and need further assessment. LMS monitoring in crabs is a sensitive tool for evaluating exposure to concentrations of selected drugs under laboratory conditions and provides a robust tier 1 testing approach (screening biomarker) for rapid assessment of marine pollution and environmental impact assessments for analyzing pharmaceutical contamination in aquatic environments. © 2012 Springer Science+Business Media B.V.


Aguirre-Martinez G.V.,University of Cádiz | Aguirre-Martinez G.V.,Campus Universitario Of Puerto Real | Buratti S.,University of Bologna | Fabbri E.,University of Bologna | And 3 more authors.
Journal of Environmental Sciences (China) | Year: 2013

Although pharmaceuticals have been detected in the environment only in the range from ng/L to μg/L, it has been demonstrated that they can adversely affect the health status of aquatic organisms. Lysosomal membrane stability (LMS) has previously been applied as an indicator of cellular well-being to determine health status in bivalve mussels. The objective of this study is to evaluate LMS in Ruditapes philippinarum haemolymph using the neutral red retention assay (NRRA). Clams were exposed in laboratory conditions to caffeine (0.1, 5, 15, 50 μg/L), ibuprofen (0.1, 5, 10, 50 μg/L), carbamazepine and novobiocin (both at 0.1, 1, 10, 50 μg/L) for 35 days. Results show a dose-dependent effect of the pharmaceuticals. The neutral red retention time measured at the end of the bioassay was significantly reduced by 50% after exposure to environmental concentrations (. p < 0.05) (caffeine = 15 μg/L; ibuprofen = 10 μg/L; carbamazepine = 1 μg/L and novobiocin = 1 μg/L), compared to controls. Clams exposed to these pharmaceuticals were considered to present a diminished health status (retention time < 45 min), significantly worse than controls (96 min) (. p < 0.05). The predicted no environmental effect concentration (PNEC) results showed that these pharmaceuticals are very toxic at the environmental concentrations tested. Measurement of the alteration of LMS has been found to be a sensitive technique that enables evaluation of the health status of clams after exposure to pharmaceuticals under laboratory conditions, thus representing a robust Tier-1 screening biomarker. © 2013 The Research Centre for Eco-Environmental Sciences, Chinese Academy of Sciences.


Aguirre-Martinez G.V.,University of Cádiz | Aguirre-Martinez G.V.,Campus Universitario Of Puerto Real | Del Valls T.A.,University of Cádiz | Martin-Diaz M.L.,University of Cádiz | Martin-Diaz M.L.,Campus Universitario Of Puerto Real
Ecotoxicology and Environmental Safety | Year: 2013

One of the main consequences of the constant input of pharmaceuticals to the aquatic environment is that biota might develop unknown chronic effects, thus affecting their health even at low concentrations. The aim of this study is to evaluate the health status of Carcinus maenas employing a 2-tier approach, after 28 days of exposure to carbamazepine (CBZ) and novobiocin (NOV) at 0.1, 1, 10 and 50μgL-1. Lysosomal membrane stability (LMS) is employed in tier 1. In tier 2 was applied a battery of biomarkers of exposure and effect (ethoxyresorufin O-deethylase (EROD), dibenzyl flourescein dealkylase (DBF), glutathione S-transferase (GST), glutathione peroxidase (GPx), lipid peroxidation (LPO) and DNA adducts) measured in gill, hepatopancreas, muscle and gonad tissues. Results show a dose-dependent effect. LMS in crabs exposed to environmental concentrations of pharmaceuticals was significantly lower compared to controls (p<0.05), indicating their stressed status. EROD activity was induced significantly (p<0.05) in all tissues by NOV (10-50μgL-1). DBF activity was induced significantly (p<0.05) in gill and hepatopancreas tissues by CBZ (10-50μgL-1). GST activity was activated in all tissues of crabs exposed to the highest concentrations tested (p<0.05). All tissues showed induction of GPX activity after exposure to selected drugs (p<0.05). LPO was activated in gill and hepatopancreas tissues by the pharmaceuticals at 50μgL-1 (p<0.05). Crabs exposed to NOV (50μgL-1) presented DNA damage in gill and hepatopancreas tissues (p<0.05). Environmental concentrations of these pharmaceuticals have a measurable effect on the biomarkers studied. The 2-tier approach applied might be a suitable tool for the assessment of sublethal responses in crabs exposed to pharmaceuticals in the marine environment. © 2013 Elsevier Inc.


Aguirre-Martinez G.V.,University of Cádiz | Aguirre-Martinez G.V.,Campus Universitario Of Puerto Real | Owuor M.A.,University of Cádiz | Owuor M.A.,South Eastern Kenya University | And 5 more authors.
Chemosphere | Year: 2015

Nowadays, the presence of pharmaceutical products in aquatic environments is not only common, but is also of significant concern regarding the adverse effect they may produce to aquatic biota. In order to determine the adverse effects of caffeine (CAF), ibuprofen (IBU), carbamazepine (CBZ) and novobiocin (NOV), at environmental occurring concentrations, standardized endpoints applied in current guidelines were evaluated in four organisms including bioluminescence response in Vibrio fischeri, growth inhibition in Isochrysis galbana (marine water) and Pseudokirchneriella subcapitata (fresh water) and fertilization and embryo-larval development in Paracentrotus lividus. To reach this aim bioassays were implemented by exposing organisms to water spiked with drugs dissolved in DMSO (0.001% v/v). Risk characterization was performed, calculating the environmental impact of drugs by calculating environmental concentration and predicted no effect concentration ratio (MEC/PNEC). Results indicate that acute toxicity was found above environmental concentrations in the order of mgL-1 for bacteria bioluminescence, microalgae growth inhibition and sea urchin fertilization. However, teratogenicity was observed on sea urchin after exposure to environmental concentrations of drugs at 0.00001mgL-1; at this concentration CBZ and IBU were found to reduce significantly the embryo-larval development compared to controls (p<0.01). The risk calculated for selected drugs suggested they are harmless for aquatic environment except when applying the embryo-larval development endpoint. Endpoints applied in this study showed the necessity of using more sensitive responses, when assessing risk of pharmaceuticals in aquatic environments, since endpoints applied in current guidelines may not be suitable. © 2014 Elsevier Ltd.


Carballeira C.,University of Cádiz | Ramos-Gomez J.,University of Santiago de Compostela | Martin-Diaz M.L.,University of Cádiz | Martin-Diaz M.L.,Campus Universitario Of Puerto Real | And 2 more authors.
Journal of Environmental Monitoring | Year: 2012

The increase in aquaculture activities in the last few decades has not been accompanied by a corresponding increase in environmental controls and regulations. In this context, the application of environmental monitoring plans (EMPs) has become necessary to assess the environmental impact associated with fish farming wastes. The objective of this review paper is to evaluate the suitability of experimental and analytical procedures as monitoring tools for inclusion in EMPs for intensive land-based marine fish farms (LBMFFs). The strong hydrodynamics and, in particular, the lack of sediment on the rocky coasts where LBMFFs are usually located, greatly limit the monitoring tools that can be used. We propose EMPs that employ a weight-of-evidence approach to evaluate: contamination, trophic and toxic effects, and ecological integrity. Laboratory tests, in situ bioassays and field surveys of local species are presented as key tools for assessing the impact of LBMFFs on ecosystems. The δ15N signal along a spatial gradient is proposed for evaluating exposure to contaminants. Trophic effects can be determined by growth of transplanted macro- and microalgae. Toxic effects can be evaluated by responses at different levels of biological organization, including biochemical and histological changes, physiological alterations and survival, in species from different trophic levels. Fouling tests and analysis of community structures are recommended for assessing ecological integrity. This review contributes to the development of environmental controls for intensive LBMFFs, and for other activities that discharge wastewater to rocky shores. © 2012 The Royal Society of Chemistry.

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