Characterization and in vitro sensitivity of cholinesterases of gilthead seabream (Sparus aurata) to organophosphate pesticides.

The characterization of cholinesterase activity in brain and muscle of gilthead seabream was carried out using four specific substrates and three selective inhibitors. In addition, K m and V max were calculated from the Michaelis-Menten equation for ASCh and BSCh substrates. Finally, the in vitro sensitivity of brain and muscle cholinesterases to three organophosphates (OPs) was also investigated by estimating inhibition kinetics. The results indicate that AChE is the enzyme present in the brain, whereas in muscle, a typical AChE form is present along with an atypical form of BChE. Very low ChE activity was found in plasma with all substrates used. The inhibitory potency of the studied OPs on brain and muscle AChEs based on bimolecular inhibition constants (k i ) was: omethoate < dichlorvos < azinphosmethyl-oxon. Furthermore, muscle BChE was found to be several orders of magnitude (from 2 to 4) more sensitive than brain and muscle AChE inhibition by dichlorvos and omethoate.

Effect of two sulfur-containing amino acids, taurine and hypotaurine in European sea bass (Dicentrarchus labrax) sperm cryopreservation.

In the present work, taurine and hypotaurine were evaluated as potential additives to improve European sea bass (Dicentrarchus labrax) sperm quality after cryopreservation. For cryopreservation, three different extenders were used: control extender (NAM), supplemented with 1mM taurine or supplemented with 1mM hypotaurine, all of them containing 10% Me2SO as cryoprotectant. To evaluate sperm quality of fresh and thawed sperm, motility (CASA: computer assisted sperm analysis), viability (SYBR Green/propidium iodide), lipid peroxidation (malondialdehyde level), protein oxidation (carbonyl content), glutathione peroxidase, glutathione reductase and superoxide dismutase activities and DNA fragmentation (comet assay) were quantified. The result demonstrated that 1 mM hypotaurine supplemented extender increased total motility (30.1 ± 3.2%), and that 1 mM taurine extender produced higher velocity (18.1 ± 2.6 µm/s) and linearity (46.0 ± 4.8%) than the control extender (21.8 ± 3.2%, 15.5 ± 1.3 µm/s, 41.8 ± 2.4%, respectively). Cell viability, lipid peroxidation and protein oxidation were not statistically different between treatments. Similar results were obtained for glutathione peroxidase and superoxide dismutase activities. Only glutathione reductase showed differential activity before and after freezing, increasing its activity in thawed sperm. Regarding the comet assay results, taurine and hypotaurine significantly reduced DNA fragmentation (52.8 ± 0.9% and 51.8 ± 0.9%, respectively) in comparison to the control (55.7 ± 0.8%). In conclusion, for European sea bass sperm cryopreservation, extenders supplemented with 1 mM taurine and 1 mM hypotaurine improved some parameters of sperm quality after thawing, resulting in better motility and lower DNA damage than the control, two very important factors related to fertilization success.

Antiviral activity of casein and αs2 casein hydrolysates against the infectious haematopoietic necrosis virus, a rhabdovirus from salmonid fish.

Salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), are responsible for serious losses in the rainbow trout and salmon-farming industries, and they have been the subject of intense research in the field of aquaculture. Thus, the aim of this work is to study the antiviral effect of milk-derived proteins as bovine caseins or casein-derived peptides at different stages during the course of IHNV infection. The results indicate that the 3-h fraction of casein and α(S2) -casein hydrolysates reduced the yield of infectious IHNV in a dose-dependent manner and impaired the production of IHNV-specific antigens. Hydrolysates of total casein and α(S2) -casein target the initial and later stages of viral infection, as demonstrated by the reduction in the infective titre observed throughout multiple stages and cycles. In vivo, more than 50% protection was observed in the casein-treated fish, and the kidney sections exhibited none of the histopathological characteristics of IHNV infection. The active fractions from casein were identified, as well as one of the individual IHNV-inhibiting peptides. Further studies will be required to determine which other peptides possess this activity. These findings provide a basis for future investigations on the efficacy of these compounds in treating other viral diseases in farmed fish and to elucidate the underlying molecular mechanisms of action. However, the present results provide convincing evidence in support of a role for several milk casein fractions as suitable candidates to prevent and treat some fish viral infections.

Transmission of lymphocystis disease virus to cultured gilthead seabream, Sparus aurata L., larvae.

The transmission of lymphocystis disease virus (LCDV) to gilthead seabream, Sparus aurata L., larvae was investigated using fertilized eggs from a farm with previous reports of lymphocystis disease. LCDV genome was detected by PCR-hybridization in blood samples from 17.5% of the asymptomatic gilthead seabream broodstock analysed. Using the same methodology, eggs spawned from these animals were LCDV positive, as well as larvae hatched from them. The presence of infective viral particles was confirmed by cytopathic effects development on SAF-1 cells. Whole-mount in situ hybridization (ISH) and immunohistochemistry (IHC) showed the presence of LCDV in the epidermis of larvae hatched from LCDV-positive eggs. When fertilized eggs were disinfected with iodine, no viral DNA was detected either in eggs (analysed by PCR-hybridization) or in larvae (PCR-hybridization and ISH). These results suggest the vertical transmission of LCDV, the virus being transmitted on the egg surface. Larvae hatched from disinfected eggs remain LCDV negative during the endotrophic phase, as showed by PCR-hybridization, ISH and IHC. After feeding on LCDV-positive rotifers, viral antigens were observed in the digestive tract, which suggests that viral entry could be achieved via the alimentary canal, and that rotifers can act as a vector in LCDV transmission to gilthead seabream larvae.

Application of in situ detection techniques to determine the systemic condition of lymphocystis disease virus infection in cultured gilt-head seabream, Sparus aurata L.

Immunohistochemistry (IHC) and in situ hybridization (ISH) techniques have been used for the detection of lymphocystis disease virus (LCDV) in formalin-fixed, paraffin-embedded tissues from gilt-head seabream, Sparus aurata L. Diseased and recovered fish from the same population were analysed. IHC was performed with a polyclonal antibody against a 60-kDa viral protein. A specific digoxigenin-labelled probe, obtained by PCR amplification of a 270-bp fragment of the gene coding the LCDV major capsid protein, was used for ISH. LCDV was detected in skin dermis and gill lamellae, as well as in several internal organs such as the intestine, liver, spleen and kidney using both techniques. Fibroblasts, hepatocytes and macrophages seem to be target cells for virus replication. The presence of lymphocystis cells in the dermis of the skin and caudal fin, and necrotic changes in the epithelium of proximal renal tubules were the only histological alterations observed in fish showing signs of the disease.

Comparative gene expression of gonadotropins (FSH and LH) and peptide levels of gonadotropin-releasing hormones (GnRHs) in the pituitary of wild and cultured Senegalese sole (Solea senegalensis) broodstocks.

The Senegalese sole (Solea senegalensis) is a valuable flatfish for aquaculture, but it presents important reproductive problems in captivity. Spawning is achieved by wild-caught breeders but cultured broodstocks fail to spawn spontaneously and, when they do, eggs are unfertilized. To gain knowledge on the physiological basis underlying this reproductive dysfunction, this study aimed at analyzing comparative hormone levels between wild and cultured broodstocks at the spawning season. The Senegalese sole gonadotropin (GTH) subunits, FSHbeta, LHbeta and GPalpha, were cloned and qualitative (in situ hybridization) and quantitative (real-time PCR) assays developed to analyze pituitary GTH gene expression. In females, FSHbeta and GPalpha mRNA levels were higher in wild than in cultured broodstocks, whereas in males all three subunits were highest in cultured. By ELISA, three GnRH forms were detected in the pituitary, displaying a relative abundance of GnRH2>GnRH1>GnRH3. All GnRHs were slightly more abundant in wild than cultured females, whereas no differences were observed in males. Plasma levels of vitellogenin and sex steroids were also analyzed. Results showed endocrine differences between wild and cultured broodstocks at the spawning period, which could be related to the endocrine failure of the reproductive axis in cultured breeders.

The Bromodomain testis-specific gene (Brdt) characterization and expression in gilthead seabream, Sparus aurata, and European seabass, Dicentrarchus labrax.

Multiple genes and transcription factors are involved in regulation and control of the complex process of sex determination and differentiation of fish species. Also more, several hormonal factors and some environmental conditions can also be adequate spawning strategies and stimuli for inducing reproduction of fish species. Brdt gene belongs to the bromodomain-extraterminal domain (BET) family of transcriptional coregulators. In mammals, Brdt gene is almost exclusively expressed in testis. Furthermore, Brdt protein is involved in elongating spermatids, and is required for proper spermatogenesis and male fertility. However, from our understanding of fish species, the role of this gene as key, during gametogenesis, still remains unknown. In this study, two Brdt mRNA transcripts were isolated from two teleostean fish species, gilthead seabream and European seabass. In both species the shorter form lacked a functional C-terminal domain, which may involve a different function as transcriptional regulator. The pattern of Brdt expression showed that the highest levels occurred in the gonads. Significantly lower levels of expression were detected in brain, pituitary and different organ systems (heart, kidney, gills, among other somatic tissues) from both studied species. In situ hybridization approach evidenced that Brdt mRNA expression was restricted to specific cell-types of the germ line, during both oogenesis and spermatogenesis processes.

Male reproductive system in Senegalese sole Solea senegalensis (Kaup): anatomy, histology and histochemistry.

Despite the fact that the Senegalese sole Solea senegalensis is a target species for the aquaculture industry in Spain and Portugal, very little is known about its gametogenesis and especially about testicular development. Therefore, the male reproductive system in adult S. senegalensis was described using a histological and histochemical approach. Mean gonadosomatic index was very low (0.094+/-0.004%) and suffered slight changes throughout the experimental period. In transverse sections, the testis presents a reniform structure with two main regions, a cortical one with seminiferous lobules where germ cells proliferate in spermatocysts (germ/Sertoli cells units), and a medullar one with spermatic ducts that collect and store the produced sperm. The germinal compartment is organized into branching lobules of the unrestricted spermatogonial type, although the majority of type A spermatogonia are located at the distal part of the lobules. Spermatogenesis seems to be semi-cystic, since spermatids are released into the lobule lumen, where they are transformed into spermatozoa. Proteins in general, especially those rich in arginine, and carboxylated mucosubstances/glyco-proteins increased from spermatogonia to spermatozoa. 3beta-Hydroxysteroid dehydrogenase enzymatic activity was exclusively observed in Leydig cells. The present study provides the first precise description of male reproductive apparatus in S. senegalensis.

Cellular distribution and induction of CYP1A following exposure of gilthead seabream, Sparus aurata, to waterborne and dietary benzo(a)pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin: an immunohistochemical approach.

The present study aimed to investigate cellular expression of cytochrome P4501A (CYP1A) protein in the seabream, Sparus aurata, exposed to one of two CYP1A-inducing contaminants, benzo(a)pyrene (B(a)P) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Male adult fish were exposed to several concentrations of TCDD or B(a)P either via water or via food. Fish were sampled after 0, 5, 10, 15 or 20 days of treatment and the time- and concentration-dependent induction of CYP1A protein in cells and tissues was studied using immunohistochemistry. A general site of CYP1A induction was the vascular endothelium. Aqueous exposures resulted in elevation of CYP1A immunoreactivity in gill pillar cells, heart endothelium, renal tubular epithelium, hepatocytes, and gut mucosal epithelium. In contrast, dietary exposure resulted in strong CYP1A immunostaining in gut epithelium but in only mild to moderate staining elsewhere. Both B(a)P and TCDD induced CYP1A in similar cellular response patterns in most organs examined, although TCDD generally led to higher staining intensity and frequency (i.e. the number of CYP1A-positive cells within an organ), an effect that is likely to be related to compound-specific differences in induction potency, metabolism and penetration. Contaminant-specific staining patterns were observed in the gills, where TCDD exposure evoked CYP1A immunostaining in the endothelial pillar cells, while B(a)P induced CYP1A staining in the branchial epithelial cells. This work points to the importance of immunohistochemical identification of cell-specific CYP1A responses in assessing the toxicology of CYP1A-inducing xenobiotics.

New tetrachromic VOF stain (Type III-G.S) for normal and pathological fish tissues.

A new VOF Type III-G.S stain was applied to histological sections of different organs and tissues of healthy and pathological larvae, juvenile and adult fish species (Solea senegalensis; Sparus aurata; Diplodus sargo; Pagrus auriga; Argyrosomus regius and Halobatrachus didactylus). In comparison to the original Gutiérrez VOF stain, more acid dyes of contrasting colours and polychromatic/metachromatic properties were incorporated as essential constituents of the tetrachromic VOF stain. This facilitates the selective staining of different basic tissues and improves the morphological analysis of histochemical approaches of the cell components. The VOF Type III -6.5 stain is composed of a mixture of several dyes of varying size and molecular weight (Orange G

Distribution of serotonin in the brain of the Senegalese sole, Solea senegalensis: an immunohistochemical study.

We report the distribution of serotonin immunoreactive (5-HT-ir) structures in the brain of the adult Senegalese sole, Solea senegalensis, using the streptavidin-biotin-peroxidase complex immunohistochemical method. We have found a wide distribution of immunoreactive fibers throughout the entire brain. 5-HT-ir cell bodies appeared restricted to some periventricular nuclei associated with the diencephalic recesses, and in the rhombencephalic reticular formation and inferior olivary region. Specifically, cerebrospinal fluid-contacting serotoninergic cells were found within the pars dorsalis and pars ventralis of the nucleus recessus lateralis, in the paraventricular organ and in the nucleus recessus posterioris. In the brainstem, 5-HT-ir perikarya appear within the superior and inferior raphe, the nucleus reticularis superioris, the nucleus interpeduncularis and the inferior olive. Although positive fibers were not found in the neurohypophysis, a few 5-HT-ir cells were identified in the adenohypophysis. This distribution is compared with those found in other fishes and discussed in the context of putative roles of 5-HT as a neuroendocrine factor and neurotransmitter in the Senegalese sole.

Localization of tyrosine hydroxylase-immunoreactivity in the brain of the Senegalese sole, Solea senegalensis.

The localization of catecholamines in the brain of the Senegalese sole was determined by immunohistochemical techniques using antibodies against tyrosine hydroxylase. Although the general pattern of distribution of catecholamines is consistent with that reported in other teleosts, some remarkable differences are observed. The most rostral tyrosine hydroxylase immunoreactive (TH-ir) cells were identified in the olfactory bulbs, in which a clear asymmetry in the number and location of TH-ir perikarya and fibers was observed. The number of TH-ir cells is manifestly higher in the right olfactory bulb, especially in the internal cell layer. TH-ir fibers are also much more abundant in the right bulb, principally in the glomerular and internal cell layers. Other TH-ir cell masses were identified in the ventral telencephalon, preoptic area, caudoventral hypothalamus, posterior tuberculum, synencephalon, isthmic region and rhombencephalon. Surprisingly, no ir cell bodies were identified in the ventromedial thalamic nucleus, which exhibits a large number of TH-ir cells in other teleosts. The presence of TH-ir fibers in the brain of sole is particularly evident within and around the nuclei in which immunoreactive cells are found. However, other zones such as the dorsal telencephalon, posterior commissure, optic tectum, torus semicircularis, reticular formation or inferior olive also displayed TH-ir fibers. TH-ir axons also enter the infundibulum, reaching the proximal pars distalis of the adenohypophysis. The distribution of TH-ir cells and fibers is compared with that observed in other teleosts and is discussed in a comparative context.

The torus longitudinalis in the gilthead seabream: an undescribed fiber tract link with the valvula cerebelli.

In this paper, we present a brief anatomical description of the torus longitudinalis and the valvula cerebelli in a percomorph fish, the gilthead seabream (Sparus aurata) based on the analysis of serial brain sections stained by paraldehyde fuchsin, Groat's hematoxylin-picroindigocarmin and cresyl violet. The existence of a small undescribed fiber tract directly bridging the ventral torus longitudinalis and the granular layer of the rostral valvula cerebelli is also reported. This small fiber tract was observed in its integrity on a few transverse sections providing that the angle of sectioning was appropriate. The existence of the anatomical link between the TL and the VC described in this paper might sustain the role of the TL as part of an ascending cerebello-tectal circuit, at least in gilthead seabream. However, these fibers might also represent fibers of passage originating from other brain structures.

Histological and histochemical observations in the stomach of the Senegal sole, Solea senegalensis.

An histological and histochemical study was conducted on the stomach of adult Senegal sole, Solea senegalensis specimens. The stomach was made up of four distinct layers: mucosa, lamina propria-submucosa-, muscularis and serosa. Surface epithelial, glandular and rodlet cells were present in the mucosa. Cells of the columnar epithelium contained a basal nucleus. Numerous mitochondria, granular endoplasmic reticulum and Golgi apparatus consisting of several parallel cisternae and vesicles were observed in the cytoplasm of these cells. The lysosomes were small, round and dense. The gastric glands were numerous in the pyloric and fundic regions but absent in the cardiac stomach. These glands were formed by two cell-types: light and dark cells. The light cells were characterised by numerous mitochondria, while dark cells had slightly fewer mitochondria and a tubulo-vesicular system. Rodlet cells similar to those observed in other teleostean fish were present among the epithelial cells. Although the epithelial cells of the mucosa contained a weak presence of neutral and acid mucopolysaccharides/mucosubstances, these substances were abundant in the lamina propria-submucosa. Proteins rich in arginine, lysine, cysteine and cystine were rarely present in the mucosa and lamina propria-submucosa of stomach, while proteins rich in tyrosine were abundant in these layers. Acid phosphatase, and ATP-ase (pH 7.2 and 9.4) activities were detected in the mucosa and lamina propria-submucosa. Alkaline phosphatase activity was not detected.

A morphological study of the brain of Solea senegalensis. I. The telencephalon.

In this paper we present an anatomical description of the telencephalon of Solea senegalensis based on cresyl violet and haematoxilin-eosin-stained serial transverse sections. This work was conducted as a basis for the precise localization of neuroendocrine territories in the brain of a species with growing interest in marine aquaculture. The external asymmetric morphology of Senegalese sole is correlated with the asymmetry of the forebrain. The right olfactory nerve and bulb are larger than the contralateral ones and this asymmetry is also extended to the cerebral hemispheres. The olfactory bulb comprises an outer olfactory nerve fiber layer, a glomerular layer, an external cellular layer, a secondary olfactory fiber layer and an internal cellular layer. The telencephalic hemispheres can be divided in area dorsalis and area ventralis, consisting of eleven and eight cell masses, respectively. The area dorsalis comprises five subareas: a pars medialis (Dm), subdivided into four nuclei termed Dml to Dm4; a pars dorsalis (Dd); a pars lateralis (D1), which consists of dorsal (Dld), ventral (Dlv) and posterior (Dlp) subdivisions; a pars centralis (Dc); and more caudally, a pars posterioris (Dp), which is very prominent in this species. A nucleus taenia (NT) was observed in the transitional region between area dorsalis and area ventralis. The area ventralis consists of pars dorsalis (Vd), pars ventralis (Vv), pars supracommissuralis (Vs), pars postcommissuralis (Vp), pars lateralis (V1), pars centralis (Vc), pars intermedia (Vi) and nucleus entopeduncularis (E). A periventricular organ, that we have termed lateral septal organ (LSO), was observed in the ventral telencephalon, medial to Vv.

Oogenesis in the bluefin tuna, Thunnus thynnus L.: a histological and histochemical study.

Histology and histochemistry are useful tools to study reproductive mechanisms in fish and they have been applied in this study. In the bluefin tuna, Thunnus thymus L., oocyte development can be divided into 4 principal phases based on the morphological features of developing oocytes and follicles. The primary growth phase includes oogonia and basophilic or previtellogenic oocytes classified as chromatin-nucleolus and perinucleolus stages. The secondary growth phase is represented by vitellogenic oocytes at early (lipid globule and yolk granule 1), mid (yolk granule 2) and late (yolk granule 3) vitellogenesis stages. The maturation phase involves postvitellogenic oocytes undergoing maturation process. During the spawning period, both postovulatory follicles, which indicate spawning, and atretic follicles can be distinguished in the ovary. Carbohydrates, lipids, proteins and specially those rich in tyrosine, tryptophan, cystine, arginine, lysine and cysteine, as well phospholipids and/or glycolipids and neutral glycoproteins were detected in yolk granules. Moreover, affinity for different lectins (ConA, WGA, DBA and UEA) was detected in vitellogenic oocytes (yolk granules, cortical alveoli, follicular layer and zona radiata), indicating the presence of glycoconjugates with different sugar residues (Mannose- Man- and/or Glucose -Glc-; N-acetyl-D-glucosamine- GlcNAc- and/or sialic acid- NANA-; N-acetyl-D-galactosamine- GalNAc-; L-Fucose -Fuc-). Histochemical techniques also demonstrated the presence of neutral lipids in globules (vacuoles in paraffin sections) and neutral and carboxylated mucosubstances in cortical alveoli. By using anti-vitellogenin (VTG) serum, immunohistochemical positive results were demonstrated in yolk granules, granular cytoplasm and follicular cells of vitellogenic oocytes. Calcium was also detected in yolk granules and weakly in follicular envelope. In females, the gonadosomatic index (GSI) increased progressively from May, during early vitellogenesis, until June during mid and late vitellogenesis, where the highest values were reached. Subsequently, throughout the maturation-spawning phases (July), GSI decreased progressively reaching the minimal values during recovering-resting period (October).

Organogenesis of the digestive tract in the white seabream, Diplodus sargus. Histological and histochemical approaches.

The ontogeny of the digestive tract of the white seabream, Diplodus sargus during the larval development up to day 45 post-hatching (dph) has been studied using histological and histochemical techniques. The oesophageal goblet cells appeared around 6 dph and contained neutral and acid mucosubstances (PAS/diastase-PAS and Alcian Blue pH 2.5 positive reactions). An incipient stomach can be distinguished from 2 dph but the first sign of gastric gland development was detected around 13-15 dph, increasing in number and size by 22-23 dph. Gastric glands were concentrated in the cardiac stomach region and they had a high content of protein rich in tyrosine, arginine and tryptophan. Acidophilic supranuclear inclusions related to pynocitosis of proteins, were already observed in the intestinal cells of the posterior intestine around 4-6 dph (exogenous feeding) and they were present until 25 dph. The intestinal mucous cells appeared between 15-18 dph and contained a mixture of neutral and acid mucosubstances/glycoconjugates, carboxylated ones being more abundant than the sulphated ones. The stomach and gastric glands were fully developed by the first month of life marking the beginning of digestive features characteristic of the juvenile stage. Around 4-6 dph, glycogen, proteins and neutral lipids were observed in the granular cytoplasm of hepatocytes. Strongly acidophilic zymogen granules were also present, at this time, in the basophilic cytoplasm of the exocrine pancreatic acinar cells and contained abundant proteins, especially rich in arginine, tyrosine and tryptophan.