Biochemical effects and polycyclic aromatic hydrocarbons (PAHs) in senegal sole (Solea senegalensis) from a Huelva estuary (SW Spain).

Relations between several stress oxidative biomarkers and aromatic polycyclic hydrocarbon (PAH) concentrations have been studied in wild sole, Solea senegalensis collected in the vicinity of a petrochemical industry. Antioxidant enzyme activities in eco-toxicological studies constitute excellent markers for exposure to a large variety of pollutants. The 16 PAHs in sediment as well as oxidative damage (LPO), activity of catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR) and PAHs type metabolites in sole liver were analysed. Significant correlations (p<0.05) were established between some biomarkers as GST, GPx and CAT and PAHs metabolites in liver (naphthalene, pyrene and phenanthrene) and PAHs concentrations in sediments (fluoranthene, acenaphthene, anthracene and chrysene). PAHs accumulated in the sediment and organisms are inducers of antioxidant defences. GST, GPx and CAT were robust biomarkers showing correlations with both PAHs in sediments and liver PAH metabolites showing different responses to low and high molecular weight PAHs.

Sublethal and lethal toxicity in juvenile Senegal sole (Solea senegalensis) exposed to copper: a preliminary toxicity range-finding test.

Static bioassay test for acute toxicity of copper in Senegal sole juveniles (Solea senegalensis) was conducted. The 96h LC(50) value of metal was found to be 0.32mg/L Cu. The intensity of histological alterations was increased gradually with the copper concentration and the exposure time. Numerous aneurysms, hypertrophy, hyperplasia and fusion of the lamellar epithelium in gills and a disorganization of the hepatic parenchyma and vacuolization in liver were common lesions in S. senegalensis juveniles exposed to copper. The results of the study indicate the sensitivity of juvenile S. senegalensis to copper (II), the persistence of sublethal effects and histology as a tool capable of revealing the sublethal effects of heavy metals on the environment and aquatic biota.

Development of the Senegal sole, Solea senegalensis forebrain.

The present paper deals with the ontogeny of the forebrain of the flatfish Senegal sole, Solea senegalensis, through different developmental stages before and after to metamorphosis. A first approach was made by conventional histological techniques, which allowed the determination of the main ontogenetic events. A second approach was to analyze the proliferation zones (PZ) during development and their locations, as well as the relation between them and the telencephalic asymmetry of the Senegal sole. The results show that before metamorphosis the Senegal sole goes through a fast development. The pituitary is visible 1 day after hatching (DAH), the inferior lobes of the hypothalamus appear 3 DAH, and the olfactory bulb and the differentiation between telencephalon and diencephalon are present around 4 DAH. In addition, by applying proliferating cell nuclear antigen (PCNA) immunohistochemistry by means of a monoclonal antibody against the PCNA and ABC complex, we were able to determine the PZs in the forebrain of pre- and post- metamorphic specimens. Although in both cases the PZs were similar, in premetamorphic animals they were thicker. However, PZs were observed in the pallium and subpallium, preoptic region, pretectum, epithalamus, dorsal and ventral thalamus, posterior tuberculum and hypothalamus. In all cases the PZs, mainly focusing on the telencephalon, were symmetrical in both hemispheres.

Expression, cellular distribution and induction of cytochrome p4501A (CYP1A) in gilthead seabream, Sparus aurata, brain.

The presence and induction of cytochrome p4501A (CYP1A) in the brain of a teleost fish, the seabream, Sparus aurata, was studied. Cerebral CYP1A expression of control fish or fish exposed to various concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was assessed at the enzyme activity level-measured as 7-ethoxyresorufin-O-deethylase; at the protein level-measured by means of Western blot and ELISA; and at the mRNA level-estimated by means of RT-PCR. Cellular localization of CYP1A in the brain tissue was studied using immunohistochemistry. In non-exposed control fish, expression of CYP1A could be demonstrated only in the olfactory bulbs. After TCDD exposure, the olfactory bulbs still showed the highest expression levels of CYP1A, however, other brain regions were now CYP1A-positive as well. Immunohistochemical examination of brain tissue sections from control fish demonstrated CYP1A immunoreactive fibers in the ventral telencephalon, in the glomerular layer of the olfactory bulbs, and in the endothelia of the cerebral vascular system. The same structures reacted positive in TCDD-exposed fish, but cell bodies and fibers from additional brain areas including telencephalon, diencephalon, mesencephalon and cerebellum showed CYP1A immunostaining. In the pituitary of TCDD-treated fish, putative GTH cells were positive for CYP1A, whereas in control fish no staining of the adenohypophysis was observed. The present findings provide evidence for basal expression of CYP1A in the telencephalon of Sparus aurata, and for the presence of inducible CYP1A in all other major brain regions, including the pituitary.

Histopathological alterations and induction of cytochrome P-450 1A in the liver and gills of the gilthead seabream (Sparus aurata) exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated in the seabream Sparus aurata specimens. Liver presented hepatocytic alterations, with an increase of lipid droplets and glycogen granules. Ultrastructural modifications of hepatocytes included RER fractionation, glycogen augmentation, as well as a rise in the number of lipid droplets, vacuoles and secondary lysosomes. In the gills, secondary lamellar epithelium showed hyperplasia, hypertrophy and lamellar fusion on the edge of the filaments. At the end of the exposure period (1 pg1(-1) TCDD for 20 days), some organelles in epithelial cells of the secondary lamellae and the tubular system of the chloride cells appeared altered. In the liver of TCDD-exposed specimens, immunoreactive cytochrome P-450 1A was concentrated close to the cytoplasmic and nuclear membranes, and positive granules were also evident throughout cytoplasm of the hepatocytes. Significant cytochrome P-450 staining was especially evident in endothelium of the hepatic vascular system. At the beginning of the exposure (1 pg 1(-1) TCDD, for 5 and 10 days), cytochrome P-450 immunostaining was observed in the cytoplasm of scarce hepatic cells and after 20 days of treatment, specific immunostained cytoplasmic granules were detected in most hepatocytes. In gills of TCDD-treated specimens, pillar-endothelial cells showed a cytochrome P-450 1A immunostaining concentrated close to the base of gill filaments and dispersed through the gill lamellae. There was also significant cytochrome staining of the endothelium of the branchial vascular system. However, no cytochrome immunoreactivity was observed in epithelial-respiratory cells.

Histochemical study of skin and gills of Senegal sole, Solea senegalensis larvae and adults.

A battery of horseradish peroxidase-conjugated lectins (Con A, WGA and DBA), as well as conventional histochemical techniques (PAS, saponification, Alcian Blue pH 0.1, 1, 2.5, chlorhydric hydrolisis, neuraminidase, Bromophenol blue, Tioglycollate reduction and Ferric-ferricyanide-FeIII) were used to study the content and distribution of carbohydrates, proteins and glycoconjugate sugar residues on the skin and gills of Senegal sole, Solea senegalensis larvae and adults: During larval development of Solea senegalensis (from hatching until day 45 posthatching), epidermal sacciform, as well as branchial and epidermal chloride cells were unreactive with all cytochemical tests performed in this paper. Mucous or goblet cells of the corporal skin and gills containing strongly sulphated acid glycoproteins were evident on days 15-20 of larval development, as well as in epidermal and branchial mucous cells of adult specimens, which also contained GlcNAc and/or sialic acid. In adult specimen, the proteic content was higher in branchial mucous cells than in epidermal cells. In larvae, variable amounts of glycoproteins containing sialic acid, GlcNAc, GalNAc, Man and/or Glc residues were observed in epithelial cells and/or cuticle. GlcNAc and/or sialic acid sugar residues were only weakly detected in glycoproteins of some epidermal and branchial mucous cells of larvae by day 45, because from hatching until metamorphosis, lectin reactions (WGA, Con A and DBA) were negative in mucous cells.

Histochemical study of lymphocystis disease in skin of gilthead seabream, Sparus aurata L.

A battery of horseradish peroxidase-conjugated lectins (Con A, WGA and DBA), as well as conventional histochemical techniques (PAS, saponification, Alcian Blue pH 0.1, 1, 2.5, chlorhydric hydrolisis, sialidase, Bromophenol blue, Tioglycollate reduction and Ferric-ferricyanide-FeIII) were used to study the content and distribution of carbohydrates, proteins and glycoconjugate sugar residues on the skin and on the lymphocystis-infected cells of gilthead seabream, Sparus aurata. Variable amounts of glycoproteins containing sialic acid, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, mannose and/or glucose residues were observed in the cuticle and mucous cells of the corporal skin, tails and fins. Germinative and epithelial cells of the epidermis contained glycogen, proteins, carboxylated groups, as well as glycoproteins with mannose and/or glucose and N-acetyl-D-galactosamine residues. Hyaline capsule of the mature lymphocystis-infected cells was strongly stained with PAS, Alcian Blue (pH 0.5 and 2.5) and weakly positive with Alcian Blue (pH 1). Con A reacted with the granular cytoplasm, specially around hyaline capsule, and with the basophilic intracytoplasmic inclusions developed in mature lymphocystis-infected cells of Sparus aurata skin. These sugar residues (mannose and/or glucose), as well as N-acetyl-D-glucosamine and/or sialic acid and N-acetyl-D-galactosamine were not detected in the hyaline capsule of the lymphocystis disease.

Histochemical distribution and accumulation of trace metals in the heart of green and normal Crassostrea angulata specimens from different southwest Spanish coasts.

Histochemical distribution and quantification of trace metals (copper-Cu-, zinc-Zn- and iron-Fe-) were studied in oyster, Crassostrea angulata heart (auricle and ventricle) from different southwest Spanish coasts. In green Crassostrea angulata specimens (green coloration in gills and visceral mass), numerous brown cells (blackish-brown) were observed in the wall of the auricles and dispersed by connective tissue, where inflammatory lesions related with granular amoebocytes were observed. However, only a few brown cells (translucent ligh-brown) were detected in auricles of normal oysters. Histochemically, Fe, Cu and Zn granules were observed in the wall of the heart (auricles), in brown cells, as well as in the granular amoebocytes, which are increased in green Crassostrea specimens. Auricle brown cells reacted weakly with PAS and Alcian Blue techniques. These cells contain proteins, as well as cysteine and cystine groups; these residues (-SH and S-S) being very abundant in the heart of green specimens, specially in brown cells and in granular amoebocytes. Lipofuscin granules were detected in these brown cells and in granular amoebocytes. On the other hand, in the heart (normal and green specimens), Zn levels were higher than Fe and Cu concentrations and heavy metals analyzed were lower in heart of normal than in green specimens. In green specimens, Cu and Zn levels were higher in auricles than in ventricles.

Histochemical aspects of the yolk-sac and digestive tract of larvae of the Senegal sole, Solea senegalensis (Kaup, 1858).

Histochemical distribution of glycoproteins, carbohydrates and proteins rich in different amino acids were studied using histological and histochemical procedures, in Senegal sole, Solea senegalensis (Kaup, 1858) larvae from hatching until day 15. Glycogen, proteins and glycoproteins were detected in the yolk-sac of the larvae at hatching and during the yolk-resorption. The epithelial digestive system (brush border, enterocytes and goblet cells) contained neutral and acid mucins (carboxylated and/or sulphated). Glycogen was observed in the cytoplasm of the digestive absortive cells (enterocytes) and in the liver (hepatocytes) on day 3-4 posthatching. Protein reactions, and specially those that showed proteins rich in arginine, tyrosine and tryptophan, were very intense in the zymogen granules of the pancreatic cells. Oesophageal and intestinal goblet cells contained glucose N-acetyl and sialic acid residues, but the mucin content of these mucous cells did not show affinity towards Con-A, suggesting the absence of glycoproteins with Mannose and/or glucose residues. WGA showed a very intense positivity in the microvilli of the digestive epithelium of the larvae and positive granules for both lectins, specially for Con-A, were detected in the cytoplasm of the anterior intestinal enterocytes.

Histological and histochemical characteristics of the lymphocystis disease in gilthead seabream, Sparus aurata L. from the south-atlantic coast of Spain.

Lymphocystis disease of the gilthead seabream, Sparus aurata from the south Atlantic coasts of Spain was studied using various cytochemical methods for nucleic acids, proteins, carbohydrates and lipids. In lymphocystis infected cells, cytoplasm and nucleoli contained RNA. DNA was restricted to the periphery of the nucleus and within the intracytoplasmatic inclusions. During the development of infected cells, proteins rich in different aminoacids were observed in the granular cytoplasm, nucleus/nucleoli, intracytoplasm inclusions and hyaline capsule. Some glycogen was observed in the cytoplasm of these cells. The intracytoplasm inclusions and the hyaline capsule also contain hydrophilic lipids, being noticeable the presence of proteins containing S-S groups. Sulphated sialoglycoproteins and glycolipids and/or phospholipids were also components of the hyaline capsule.

A histochemical and immunohistochemical study of digestive enzymes and hormones during the larval development of the sea bream, Sparus aurata L.

The distribution of different hydrolytic enzymes and the localization of the hormones which regulate glucose metabolism during development of the digestive tract of the sea bream, Sparus aurata L., were studied. The yolk sac contains trypsin, glucose-6-phosphatase, ATPases and acid and alkaline phosphatase activities. Positive insulin, glucagon and somatostatin cells were observed in the pancreas and in the lumen of the intestinal tract during endogenous feeding. From hatching until 3 days later, the digestive tract of sea bream larvae shows no enzymatic activities. During exogenous feeding, the activities of the phosphatases and trypsin generally increase, as do the amounts of the hydrolytic enzymes and trypsin, as well as the pancreatic and intestinal hormones. The enzymatic activities gradually decrease from the anterior part towards the posterior part of the digestive tract.

New staining methods for semithin sections of tissues of Seriola dumerili embedded in methacrylate.

Semithin sections of different tissues of Seriola dumerili R. Embedded in glycol methacrylate, were stained with two staining methods normally used in conventional haematology and histology: Gutierrez's cytopanchrome and Gutierrez's polychrome V.O.F. The results observed in all tissues embedded in this resin and stained with V.O.F. are satisfactory and comparable, as to staining affinity, to those obtained on paraffin-embedded tissues. Gutierrez's trichrome stain appears to be a useful tool in the study of oogenesis for detecting changes in staining according to cellular structure and maturation stage. The results observed with cytopanchrome are similar to those obtained with toluidine blue in glycol methacrylate-embedded tissues. The use of these two staining techniques, requires no previous oxidation, alkali treatments, high temperatures or removal of the embedding medium. Furthermore, both dyes can be used at the same time: in this case, cytoplasm of previtellogenic oocytes showed a stronger affinity for cytopanchrome, and were more intensely stained by V.O.F. as the vitellogenesis of Seriola dumerili progressed. Both stain, and especially the cytopanchrome one, also are available for staining the liver, gills and testis.

Carbohydrate and protein histochemistry during oogenesis in Halobatrachus didactylus (Schneider, 1801) from the Bay of Cadiz (Spain).

Histological and histochemical characteristics were studied in Halobatrachus didactylus (Schneider, 1801) during oogenesis. Three phases could be differentiated: previtellogenesis (oogonia and basophilic oocytes), vitellogenesis (yolk synthesis) and maturation-spawning. Glycogen, glycoproteins and proteins rich in certain amino acids were present in the previtellogenic as well as in the vitellogenic cytoplasm oocytes. No acid mucosubstances were detected. Three types of yolk (vesicles, vacuoles and granules) contained different types of organic reserves; granules were essentially proteic whereas globules were lipidic. Carbohydrates and proteins were present in vesicles.