Carmen Sarasquete

The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Peninsula: a practical approach

Within the frame of the 2nd Iberian Congress of Environmental Contamination and Toxicology (University of the Basque Country, Leioa, June 1998) a workshop was held about the use of biomarkers in marine pollution monitoring. Among others, the following biomarkers received special attention: metallothionein induction, acetylcholinesterase inhibition, cytochrome P450 system induction, imposex, lysosomal enlargement and lysosomal membrane destabilisation, and peroxisome proliferation. These biomarkers can be used to evaluate exposure to and effect of different contaminants (metals, organic xenobiotics and organometallic compounds) and they can be measured using different methodological approaches (biochemistry, cytochemistry, immunochemical methods based on the use of biotechnology). Before the application of a set of biomarkers in pollution monitoring programmes, well-defined protocols of Quality Assurance have to be established to allow adequate comparison of results. It is also necessary to include analysis of standard reference materials and to obtain detailed knowledge of basal values and seasonal variations of the biomarkers in various species, as well as to integrate the information obtained with the different biomarkers. Marine bivalve molluscs such as mussels are appropriate sentinel species for most of the biomarkers proposed except for the induction of the cytochrome P450 system, which should be measured in fish, and the degree of imposex, which is a biomarker of exposure to TBT specifically measured in certain gastropod molluscs. As a result of the workshop, a battery of biomarkers of contaminant exposure and effects are proposed that could be incorporated into programmes monitoring the quality of the coastal environment in the Iberian Peninsula. These measures would be undertaken in conjunction with chemical measures of contaminant burdens in selected sentinel species.

A review on the cultivation potential of Solea senegalensis in Spain and in Portugal

Abstract The sole, Solea senegalensis , is a common high-value flatfish in Southern Europe, commonly reared in extensive aquacultural production in Portugal and Spain. Research in recent years has focused on the production of larvae and juveniles of good quality. Natural spawning of broodstock in captivity has been accomplished, and is the only way viable eggs have been obtained to date. The broodstock feeding regime is based on squid ( Loligo vulgaris ), being supplemented with polychaetes ( Hediste diversicolor ) during final maturation. Temperature plays a very important role in the onset and duration of the spawning period, with egg emission stopping below 16°C. Observed duration of the spawning period has ranged from 4 to 6 months. The total daily egg collection during the spawning season ranged from 0 to 180 g for a broodstock of 15 fish. Egg fertilization rates varied between 20% and 100%, and the percentage of viable eggs had a mean of 72.1±26.5%. Variations in egg size between batches were detected, with egg size tending to decline during the spawning season. Larvae hatch with an average size of 2.4±0.1 mm total length, but this varies widely by batch. Larvae accept Artemia nauplii as first prey and reach a size of 8 mm by day 15. Metamorphosis starts on 11 days after hatching (DAH) and is completed by 19 DAH. Survival at 19 DAH range from 29% to 87%. From 19 DAH onwards fish are fed live Artemia metanauplii, and reach 16±0.8 mm on 40 DAH. Ponds stocked with unweaned juveniles produced after one year fish with 16.6±2.1 cm total length and 40.3±2.5 g wet weight, with a survival of 20%. Unweaned juveniles stocked together with Sparus aurata reached a size of 35.3±1.8 cm total length and 456.1±3.6 g wet weight after one year fish, with a survival of 8%. Epizootic mortalities due to pasteurellosis have been observed in juvenile S. senegalensis . As in other flatfish, pigmentation abnormalities as well as some malformations associated with the migration of the eye have also been observed. The optimization of the weaning and growout procedures, including the development of appropriate feeding regimes, and further studies into the causes of pigmentation abnormalities are necessary before this species can be reared intensively on a large scale.

Histological and histochemical development of the digestive system of Solea senegalensis (Kaup, 1858) larvae

Abstract The digestive system of Solea senegalensis was studied from hatching until 1 month of larval life. Histological and histochemical procedures were used to study the histomorphology, digestive enzymes, lipid, protein and carbohydrate distribution in the digestive larval tract. The major events in digestive system differentiation occurred during the early stages. At first feeding (2DAH—days after hatching) both the mouth and anus had opened and the digestive tract was differentiated in five portions: buccal–pharyngeal cavity, esophagus, incipient stomach, anterior and posterior intestine. The pancreas and liver were also differentiated at this stage. During metamorphosis (12 to 20DAH) an elongation of the digestive tract and increase in the absorption surface were observed. Gastric glands were observed around 27DAH. Alkaline and acid phosphatase, lipase and aminopeptidase activities were observed from the mouth opening onwards and increased with larval development. Proteins exhibited a similar pattern of increase being especially abundant in the intestinal epithelium and exocrine pancreas. Neutral lipids were abundant in the yolk sac (oil globule), intestinal epithelium and liver (hepatocytes). Esophageal mucous cells were rich in sulphated mucosubstances and intestinal mucous cells, intestinal epithelium and liver were rich in neutral mucosubstances. Glycogen deposition was observed after 7DAH in liver and intestinal epithelium. The analysis of data obtained in this study suggests that after 31DAH S. senegalensis larvae are capable of ingesting, digesting and absorbing nutrients, having a morphologically complete digestive tract, equipped with digestive enzymes.

Cytochrome P4501A (CYP1A) in teleostean fishes. A review of immunohistochemical studies

Cytochrome P4501A monooxygenase has an important function in the biotransformation of many xenobiotics, including polynuclear aromatic hydrocarbons, and planar organochlorine compounds. The metabolism can lead to detoxification or activation to reactive intermediates. Exposure of fish leads to a receptor-mediated induction of CYP1A gene expression. The induction response can be quantitatively analysed by means of molecular techniques (RT-PCR, Northern Blotting), immunochemical approaches (ELISA, Western Blotting), and enzymatic methods (7-ethoxyresorufin-O-deethylase, EROD) at the catalytical level. Immunohistochemical studies have provided qualitative information on cell and tissue distribution of CYP1A in teleost fish. The liver is the major organ of CYP1A activity in fish, but the enzyme is additionally expressed in numerous extrahepatic organs, including kidney, alimentary canal, heart, gills, olfactory system, gonads, brain and endocrine tissues. In many tissues, the vascular endothelia show a strong CYP1A immunoreactivity. As indicated from immunohistochemical studies with fish embryos and larvae, the typical cell and tissue distribution of CYP1A is established early during fish ontogeny.

Glyconjugates in epidermal, branchial and digestive mucous cells and gastric glands of gilthead sea bream, Sparus aurata, Senegal sole, Solea senegalensis and Siberian sturgeon, Acipenser baeri development

Epidermal, branchial and digestive mucous cells, and the gastric glands of larvae/postlarvae (from hatching until 45 days posthatching) of three fish species (two teleostean and a chondrostean) were investigated using conventional histochemical methods (periodic acid schiff -PAS-, diastase-PAS; alcian blue pH 0.5, 1 and 2.5) in order to distinguish neutral and acidic (carboxylated and sulphated) glycoconjugates, as well as bromophenol blue reaction for identification of proteins. Additionally, the presence and distribution of sugar residues in the oligosaccharide side chains of glycoconjugates were investigated using horseradish peroxidase (HPR)-conjugated lectins (Con A, DBA, WGA and UEA-I). Most mucous cells (digestive, epidermal and branchial) of Siberian sturgeon, Acipenser baeri, sea bream, Sparus aurata and Senegal sole, Solea senegalensis larvae were PAS- and alcian blue- (pH 2.5 and 0.5) positive, with small variations between organs/tissues and species. Bromophenol blue reaction (general proteins) was positive in a minority of the mucous cells, usually in those cells which were PAS-negative. Proteins rich in sulphydryl (-SH) and/or disulphide (-S-S-) groups related with the glycoprotein nature of the glycoconjugates present in mucous cells were also observed. Epidermal, branchial and digestive mucous cells of all studied larvae did not contain glycogen or lipids. Con A lectin staining was negative in all mucous cells types of sea bream and sole, but oesophageal mucous cell of sturgeon were reactive to different lectin reactions, suggesting the presence of mannose -Man- and/or glucose -Glc-, L-fucose -Fuc- ; N-acetyl-D-galactosamine -GalNAc-, as well as N-acetyl-D-glucosamine- GlcNAc - and/or sialic acid -NANA- residues. Digestive mucous cells of all studied larvae were positive to WGA and DBA lectins. Epidermal and branchial mucous cells of sea bream and sole were Con A, DBA and UEA-I unreactive. However, mucous cells of sturgeon larvae were stained with UEA-I lectin. Gastric glands appear very early in sturgeon stomach larvae development (between 5-6 days posthatching) but rather late (around 40 days) during the ontogeny of sole and sea bream larvae. These glands contain neutral glycoproteins with Man and/or Glc, Fuc, GlcNAc- and/or sialic acid and rich in GalNAc- sugar residues, as well as proteins moderately rich in arginine, and others particularly rich in tyrosine and tryptophan.

Effect of lead on ALA-D activity, metallothionein levels, and lipid peroxidation in blood, kidney, and liver of the toadfish Halobatrachus didactylus

The effects of lead (Pb) on ALA-D activity, metallothionein (MT) levels, and lipid peroxidation in liver, kidney, and blood of the toadfish Halobatrachus didactylus were investigated. A time-course experiment was performed with sampling on days 0, 2, 5, and 7 following intraperitoneal Pb injection. This indicated a rank order for lead concentration of kidney > liver > blood in fish exposed to Pb. No significant variation of ALA-D activity was observed in liver and kidney while in blood, a slight decrease of ALA-D activity was found but this was not attributed to acute metal stress. Hepatic and renal MT levels were both affected in different ways by metal uptake. The progressive decrease of MDA concentration in the liver and the lack of a clear induction in kidney suggested the hypothesis that Pb is not a good inductor of lipid peroxidation. The histological and histochemical results demonstrated degenerative effects of lead accumulation on the tissues and the activation of lysosomal responses to induced stress.

The influence of certain aminoacids and vitamins on post-thaw fish sperm motility, viability and DNA fragmentation.

During cryopreservation, dilution in the extender media reduces the seminal plasma constituents being cells more vulnerable to oxidative stress. Vitamins (C and E) and the amino acids taurine and hypotaurine are powerful antioxidants naturally present in seminal plasma. Whether their effect may improve sperm quality and reduce sperm DNA damage after cryopreservation in fish sperm still remains unclear. Thus, the aim of the present work was to analyse the effect of extender supplementation with several antioxidant components on post-thawed sperm motility, viability and DNA integrity of two commercial species, gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax). Sperm collected from ten to twelve individuals was cryopreserved in ten different extenders containing: taurine and hypotaurine (1 and 10mM), ascorbic acid (1 and 10mM), α-tocoferol (0.1 and 0.5 mM) or 1 ml/l of a commercial cell antioxidant supplement. Cell viability, motility and DNA fragmentation were determined in post-thawed samples. Addition of antioxidants (vitamins and amino acids) to D. labrax and S. aurata extenders did not significantly increase the parameters of motility (TM, PM, VCL, VSL and Lin) or viability, although 1mM taurine slightly increased the percentage of motile cells (TM) in S. aurata. DNA fragmentation (DNA in tail and Olive tail moment) in D. labrax sperm was higher in treatments containing vitamins than amino acids or control. However in S. aurata sperm, antioxidants especially taurine and hypotaurine, significantly reduced both DNA fragmentation parameters, protecting DNA against strand breaks. These results suggest a species-specific effect depending on the type of antioxidants used.

Detection of Mineralized Structures in Early Stages of Development of Marine Teleostei Using a Modified Alcian Blue-Alizarin Red Double Staining Technique for Bone and Cartilage

We have developed a procedure for staining cartilage and bone in fish larvae as small as 2 mm (notochord length), for which standard alcian blue/alizarin red procedures did not give positive and/or consistent results. Small calcified structures only 100–200 ixm in length can be clearly visualized. The method is suitable for both onto-genic studies during early stages of skeletal development in most marine fishes (e.g., Sporus aurata L., Solea senegalensis Kaup), whose larvae at hatching are often only a few millimeters long and for detecting skeletal abnormalities in small larvae. This procedure can also be used for specimens that have been preserved in 100% ethanol for up to two years.

Substrate-SDS-PAGE determination of protease activity through larval development in sea bream

Identification of alkaline proteases produced during larval stages of gilthead sea bream, Sparus aurata was achieved using SDS-PAGE and specific inhibitors. Such techniques were also applied to determine proteases existing in rotifers, Brachionus plicatilis, and Artemia nauplii, which are used as live food for these larvae, as well as proteases of adult fish. The results show a great prominence of trypsin-like proteases during the 4 weeks after hatching, but the number of enzyme species was reduced in adult fish. Alkaline proteases present in the rotifers and Artemia showed clear differences when compared with those of the larvae and were not detected in extracts obtained from fed larvae. The results obtained provide information about the role of exogenous enzymes in larval feeding of sea bream.

Developmental expression of three different prepro-GnRH (gonadotrophin-releasing hormone) messengers in the brain of the European sea bass (Dicentrarchus labrax).

In this study, we have analyzed the ontogenic expression of three gonadotrophin-releasing hormones (GnRH) systems expressed in the brain of a perciform fish, the European sea bass, using in situ hybridization. The riboprobes used correspond to the GnRH-associated peptide (GAP) coding regions of the three prepro-GnRH cDNAs cloned from the same species: prepro-salmon GnRH, prepro-seabream GnRH and prepro-chicken GnRH II. On day 4 after hatching, the first prepro-chicken GnRH-II mRNA-expressing cells appeared in the germinal zone of the third ventricle. They increased in number and size from 10 to 21 days, reaching at day 30 their adult final position, within the synencephalic area, at the transitional zone between the diencephalon and the mesencephalon. First prepro-salmon GnRH mRNA-expressing cells became evident on day 7 arising from the olfactory placode and migrating towards the olfactory nerve. On day 10, this cell group reached the olfactory bulb, being evident in the ventral telencephalon and preoptic area from days 15 and 45, respectively. Weakly labeled prepro-seabream GnRH mRNA-expressing cells were first detected at 30 days in the olfactory area and ventral telencephalon. On day 45, prepro-seabream GnRH mRNA-expressing cells were also present in the preoptic region reaching the ventrolateral hypothalamus on day 60. The results obtained in sea bass indicate that sGnRH and sbGnRH cells have a common origin in an olfactory primordium suggesting that both forms might arise from a duplication of a single ancestral gene, while cGnRH-II cells develop from a synencephalic primordium.