Establishment and characterisation of single cell-derived embryonic stem cell lines from the gilthead seabream, Sparus aurata.

An important bottleneck in fish aquaculture research is the supply and maintenance of embryos, larvae, juvenile and adult specimens. In this context, cell lines represent alternative experimental models for in vitro studies that complement in vivo assays. This allows us to perform easier experimental design and sampling and avoid the sacrifice of animals. Embryonic stem (ES) cell lines have attracted increasing attention because they have the capability to proliferate indefinitely and could be differentiated into any cell type of the organism. To minimise cell heterogeneity and increase uniformity of in vitro studies results, in this manuscript we report the development and characterisation of two single cell-derived ES cell lines (monoclonal) from the morula stage embryos of the gilthead seabream, Sparus aurata, named as SAEC-A3 and SAEC-H7. Both cell lines have been passaged for over 100 times, indicating the establishment of long-term, immortalised ES cell cultures. Sequence analyses confirmed the seabream origin of the cell lines, and growth analyses evidenced their high viability and proliferating activity, particularly in culture medium supplemented with 10-15% fetal bovine serum and 22 °C. Both cell lines showed the ability to generate embryoid bodies and show different sensitivity and response to all-trans retinoic acid. The analysis of epithelial (col1α1) and neuronal (sox3) markers in differentiated cultures revealed that SAEC-A3 tended to differentiate towards epithelial-like cells whereas SAEC-H7 tended to differentiate towards neuronal-like cells. Both cell lines were efficiently transfected with pDsRed2-ER and/or pEGFP-N1 plasmids, indicating that they could represent useful biotechnological tools. Daily expression of pcna showed significant expression rhythms, with maximum levels of cell proliferation during the day-night transition. Currently, these cell lines are being successfully used as experimental models for the study of cellular metabolism, physiology and rhythms as well as for toxicological, pharmacological and gene expression analyses.

A universal dose-response curve for radiochromic films.

PURPOSE: This paper presents a model for dose-response curves of radiochromic films. It is based on a modified version of single-hit model to take into account the growth experienced by lithium salt of pentacosa-10,12-diynoic acid polymers after irradiation. METHODS: Polymer growth in radiochromic films is a critical phenomenon that can be properly described by means of percolation theory to provide an appropriate distribution function for polymer sizes. Resulting functional form is a power function featuring a critical exponent and two adjustable parameters. Moreover, these parameters act as scaling factors setting a natural scale for sensitometric curves where the dependence on channel sensitivity is removed. A unique reduced response curve is then obtained from all the color channels describing film behavior independently of film dosimetry system. RESULTS: Resulting functional form has been successfully tested in several sensitometric curves from different Gafchromic EBT models, providing excellent agreement with experimental data in a wide dose range up to about 40 Gy and low dose uncertainty. CONCLUSIONS: The model presented in this paper describes accurately the sensitometric curves of radiochromic films in wide dose ranges covering all typical ranges used in external radiotherapy. Resulting dose uncertainty is low enough to render a reasonably good performance in clinical applications. Due to cross-correlation, only one of the adjustable parameters is totally independent and characterizes film batches.

Metamorphosis induces a light-dependent switch in Senegalese sole (Solea senegalensis) from diurnal to nocturnal behavior.

Light plays a key role in the development of biological rhythms in fish. Recent research in Senegal sole has revealed that spawning and hatching rhythms, larval development, and growth performance are strongly influenced by lighting conditions. However, the effect of light on the daily patterns of behavior remains unexplored. Therefore, the aim of this study was to investigate the impact of different photoperiod regimes and white, blue, and red light on the activity rhythms and foraging behavior of Solea senegalensis larvae up to 40 days posthatching (DPH). To this end, eggs were collected immediately after spawning during the night and exposed to continuous white light (LL), continuous darkness (DD), or light-dark (LD) 12L:12D cycles of white (LD(W)), blue (LD(B), λ(peak) = 463 nm), or red light (LD(R), λ(peak) = 685 nm). A filming scenario was designed to video record activity rhythms during day and night times using infrared lights. The results revealed that activity rhythms in LD(B) and LD(W) changed from diurnal to nocturnal on days 9 to 10 DPH, coinciding with the onset of metamorphosis. In LD(R), sole larvae remained nocturnal throughout the experimental period, while under LL and DD, larvae failed to show any rhythm. In addition, larvae exposed to LD(B) and LD(W) had the highest prey capture success rate (LD(B) = 82.6% ± 2.0%; LD(W) = 75.1% ± 1.3%) and attack rate (LD(B) = 54.3% ± 1.9%; LD(W) = 46.9% ± 3.0%) during the light phase (ML) until 9 DPH. During metamorphosis, the attack and capture success rates in these light conditions were higher during the dark phase (MD), when they showed the same nocturnal behavioral pattern as under LD(R) conditions. These results revealed that the development of sole larvae is tightly controlled by light characteristics, underlining the importance of the natural underwater photoenvironment (LD cycles of blue wavelengths) for the normal onset of the rhythmic behavior of fish larvae during early ontogenesis.

Does lighting manipulation during incubation affect hatching rhythms and early development of sole?

Light plays a key role in the development of biological rhythms in fish. Previous research on Senegal sole has revealed that both spawning rhythms and larval development are strongly influenced by lighting conditions. However, hatching rhythms and the effect of light during incubation are as yet unexplored. Therefore, the aim of this study was to investigate the impact of the light spectrum and photoperiod on Solea senegalensis eggs and larvae until day 7 post hatching (dph). To this end, eggs were collected immediately after spawning during the night and exposed to continuous light (LL), continuous darkness (DD), or light-dark (LD) 12L:12D cycles of white light (LD(W)), blue light (LD(B); λ(peak) = 463 nm), or red light (LD(R); λ(peak) = 685 nm). Eggs exposed to LD(B) had the highest hatching rate (94.5% ± 1.9%), whereas LD(R) and DD showed the lowest hatching rate (54.4% ± 3.9% and 48.4% ± 4.2%, respectively). Under LD conditions, the hatching rhythm peaked by the end of the dark phase, but was advanced in LD(B) (zeitgeber time 8 [ZT8]; ZT0 representing the onset of darkness) in relation to LD(W) and LD(R) (ZT11). Under DD conditions, the same rhythm persisted, although with lower amplitude, whereas under LL the hatching rhythm split into two peaks (ZT8 and ZT13). From dph 4 onwards, larvae under LD(B) showed the best growth and quickest development (advanced eye pigmentation, mouth opening, and pectoral fins), whereas larvae under LD(R) and DD had the poorest performance. These results reveal that developmental rhythms at the egg stage are tightly controlled by light characteristics, underlining the importance of reproducing their natural underwater photoenvironment (LD cycles of blue wavelengths) during incubation and early larvae development of fish.

The pineal complex of the European sea bass (Dicentrarchus labrax): I. histological, immunohistochemical and qPCR study.

The pineal organ of fish is a photosensory and neuroendocrine epithalamic structure that plays a key role in the temporal organisation of physiological and behavioural processes. In this study performed in the European sea bass, Dicentrarchus labrax, we provided an in-depth description of the macroscopic and microscopic anatomy of the pineal organ and identified the presence of photoreceptor and presumed melatonin-producing cells using histological and immunohistochemical techniques. In addition, we analysed in the pineal the day-night expression (using quantitative real-time PCR) of two key enzymes in the melatonin-synthesising pathway; arylalkylamine-N-acetyltransferase 2 (AANAT2) and hydroxyindole-O-methyltransferase (HIOMT). The pineal complex of sea bass consisted of a narrow and short pineal stalk that adopts a vertical disposition, a small-sized pineal end vesicle firmly attached to the skull by connective tissue, a parapineal organ and a convoluted dorsal sac. Immunohistochemical study showed the presence of abundant serotonin-positive cells. Cone opsin-like and rod opsin-like photoreceptor cells were also evidenced in the pineal stalk and vesicle. Both Aanat2 and Hiomt were expressed in sea bass pineal organ. Aanat2 exhibited higher nocturnal transcript levels, while no significant day-night differences were found for Hiomt. These results, together with ongoing studies analysing neural and neurohormonal outputs from the pineal organ of sea bass, provide the basic framework to understand the transduction integration of light stimulus in this relevant species for marine aquaculture.

Current knowledge on the melatonin system in teleost fish.

Melatonin is a much conserved feature in vertebrates that plays a central role in the entrainment of daily and annual physiological rhythms. Investigations aiming at understanding how melatonin mediates the effects of photoperiod on crucial functions and behaviors have been very active in the last decades, particularly in mammals. In fish a clear-cut picture is still missing. Here we review the available data on (i) the sites of melatonin production in fish, (ii) the mechanisms that control its daily and annual rhythms of production and (iii) the characterization of its different receptor subtypes, their location and regulation. The in vivo and in vitro data on melatonin effects on crucial neuroendocrine regulations, including reproduction, growth, feeding and behavioral responses, are also reviewed. Finally we discuss how manipulation of the photic cues impact on fish circannual clock and annual cycle of reproduction, and how this can be used for aquaculture purposes.

Effect of calcitonin gene-related peptide (CGRP), adrenomedullin and adrenomedullin-2/intermedin on food intake in goldfish (Carassius auratus).

The purpose of the present study was to elucidate the possible role of calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin-2/intermedin (IMD) on food intake regulation in goldfish (Carassius auratus). We examined the effects of intracerebroventricular (ICV) administration of these related hormones on food intake. Food-deprived goldfish were subjected to ICV injections of CGRP, AM and IMD and their food intake were quantified. CGRP at 10ng/g body weight (bw) significantly decreased food intake as compared to saline-treated fish. IMD at 10 and 50ng/g bw both significantly decreased food intake as compared to saline group. No significant differences were observed after AM administration. Our results suggest, for the first time in fish, a role for both CGRP and IMD in the central regulation of feeding in fish.

Molecular characterization of calcitonin gene-related peptide (CGRP) related peptides (CGRP, amylin, adrenomedullin and adrenomedullin-2/intermedin) in goldfish (Carassius auratus): cloning and distribution.

To further characterize the structure and function of calcitonin gene-related peptide (CGRP) related peptides in fish, we have cloned cDNA sequences for CGRP, amylin, adrenomedullin (AM) and adrenomedullin-2/intermedin (IMD) in goldfish (Carassius auratus) and examined their tissue distribution. CGRP, amylin, AM and IMD cDNAs were isolated by reverse transcription (RT) and rapid amplification of cDNA ends (RACE). The cloned sequences contain the complete four mature peptides, which present a high degree of identity with mature peptide sequences from other fish. Phylogenetic analyses show that goldfish AM and IMD form a sub-family within the CGRP-related peptides that is distinct from the CGRP/amylin sub-family. The distribution of goldfish CGRP-like peptides mRNA expression in different tissues and within the brain was studied by RT-PCR. CGRP, IMD and AM are detected throughout the brain, in pituitary and in most peripheral tissues examined. Amylin mRNA is mostly expressed in the brain, in particular posterior brain, optic tectum and hypothalamus, but is also present in pituitary, gonad, kidney and muscle. Our results suggest that goldfish CGRP, amylin, AM and IMD are conserved peptides that show the typical structure characteristics present in their mammalian counterparts. The widespread distributions of CGRP, AM and IMD suggest that these peptides could be involved in the regulation of many diverse physiological functions in fish. Amylin mRNA distribution suggests possible new roles for this peptide in teleosts, including the control of reproduction.

Expression of gonadotrophin-releasing hormone binding sites in somatic tissues of the gilthead seabream (Sparus aurata): a quantitative autoradiographic study.

In this study, we have analysed the expression of gonadotrophin-releasing hormone (GnRH) binding sites in somatic tissues (intestine, liver, gill, skeletal muscle, ovary, heart, stomach, kidney and spleen) of the gilthead seabream, Sparus aurata using 3-[125I]iodototyrosyl5-mammalian GnRH and auto-radiographic techniques. The qualitative and quantitative analysis showed the existence of a basal expression of specific GnRH binding sites in intestine, skeletal muscle, ovary, stomach and spleen. Furthermore, our data suggest that the level of expression of GnRH binding sites can be significantly enhanced by GnRH treatment in intestine, gill, heart, stomach, kidney and spleen. This study shows that GnRH can exert direct effects in both reproductive and non-reproductive somatic tissues of the gilthead seabream.

Binding characteristics and daily rhythms of melatonin receptors are distinct in the retina and the brain areas of the European sea bass retina (Dicentrarchus labrax).

Melatonin is synthesized, with a circadian rhythm, in the pineal organ of vertebrates, high levels being produced during the scotophase and low levels during the photophase. The retina also produces melatonin, although in the case of the European sea bass, its secretion pattern appears to be inverted. In the study described here, radioreceptor assay techniques were used to characterize the melatonin binding sites, their regional distribution and their daily variations. Brain and retina membrane preparations were used in all the binding assays and 2-[125I]iodomelatonin ([125I]Mel) as radioligand at 25 degrees C. The specific binding of [125I]Mel was seen to be saturable, reversible, specific and of high affinity. In all the tissues assayed, the power of the ligands to inhibit [125I]Mel binding decreased in the following order: melatonin>>4-P-PDOT>luzindole> or =N-acetylserotonin, which points to the presence of Mel1-like receptors. The inhibition curves of 4-P-PDOT suggested the presence of two different binding sites in the brain areas, but only one type of site of low affinity in the neural retina. No daily variations in [125I]Mel binding capacity (Bmax) or affinity (Kd) were detected in the brain areas, while a clear rhythm in Kd melatonin receptor affinity and Bmax binding capacity was observed in the retina. Kd and Bmax retinal rhythms were out of phase with the lowest Kd and the highest Bmax occurring at scotophase. This result suggests that retinal melatonin is a paracrine factor able to control receptor desensitization during photophase when ocular melatonin is higher in this species.

Daily locomotor activity and melatonin rhythms in Senegal sole (Solea senegalensis).

The daily locomotor and melatonin rhythms of the Senegal sole, a benthonic species of increasing interest in aquaculture, are still unknown, despite the fact that such knowledge is of prime importance for optimising its production. The aim of the present research was therefore to investigate the daily rhythms of locomotor activity and melatonin in the Senegal sole. For this purpose, the individual locomotor activity rhythms of fish were registered using a photocell. Plasma and ocular melatonin rhythms were studied in animals reared in circular tanks placed in earth under an LD 12:12 light regime and 16-18 degrees C temperature range (spring equinox). Blood and eye samples were taken every 3 h during a complete 24-h cycle. The impact of a light pulse in the middle of the dark period (MD) on plasma melatonin was also studied. Locomotor activity was mainly nocturnal, with 84.3% of the total activity occurring during darkness. The levels of plasma melatonin were higher at night (55 pg/ml) than during the day (2 pg/ml), while ocular melatonin levels appeared to be arrhythmic. Both weight and melatonin content were found to be significantly higher in the left eye in relation to the right eye. A light pulse in MD provoked a significant decrease in plasma melatonin levels. In summary, photoperiod is a key factor in synchronizing locomotor activity and melatonin rhythms in the Senegal sole, whose nocturnal habits should be taken into account for their rearing by aquaculture.

The GnRH system in the European sea bass (Dicentrarchus labrax).

The cDNA sequences encoding three GnRH forms, sea bream GnRH (sbGnRH), salmon GnRH (sGnRH) and chicken GnRH II (cGnRH II), were cloned from the brain of European sea bass, Dicentrarchus labrax. Comparison of their deduced amino acid sequences to the same forms in the gilthead sea bream, Sparus aurata, and striped bass, Morone saxatilis, revealed high homology of the prepro-cGnRH II (94% and 98% respectively), and prepro-sGnRH (92% to both species). The sbGnRH exhibited dissimilar identities, with high homology to the striped bass (93%), and lower homology (59%) to the gilthead sea bream. Two transcript types were identified for the GnRH-associated peptide (GAP)-sGnRH as well as for the GAP-cGnRH II, which suggests a possible alternative splicing followed by the addition of an early stop codon. In order to obtain antibodies specific for the three GnRH precursors, recombinant GAP proteins were produced. The differential expression of the three GnRHs previously reported in the brain by means of in situ hybridization, using riboprobes corresponding to the GAP-coding regions, was fully confirmed by immunocytochemistry using antibodies raised against the recombinant GAP proteins, indicating that the transcripts are translated into functional proteins. Moreover, this approach allowed us to follow, for the first time, the specific projections of the different cell groups: sGAP fibers are distributed mainly in the forebrain with few projections reaching the pituitary, sbGAP fibers are mainly present in the preoptic area, mediobasal hypothalamus and predominantly project to the pars distalis of the pituitary, whereas cGnRH II fibers have a widespread distribution primarily in the posterior brain, and do not project to the pituitary. These new tools will be extremely useful to study further the development, regulation and functional significance of three independent GnRH systems in the brain of vertebrate species.

Immunocytohistochemical characterization of pituitary cells of the bluefin tuna, Thunnus thynnus L.

In this paper we report the first complete mapping of the pituitary in a tuna species. The various different adenohypophysis cell types of the bluefin tuna, Thunnus thynnus L. have been identified and located using different antisera against mammalian and piscine hormones and various histochemical techniques: PAS, Alcian Blue pH 2.5 and lectins -ConA and WGA(Neutral and Acidic Glycoproteins); Bromophenol Blue (Proteins) and Tioglycollate-Ferric-Ferricianide-FeIII (-S-S- groups). Prolactin (PRL) and adrenocorticotrophic (ACTH) cells were located in the rostral pars distalis (RPD) of the pituitary, while the proximal pars distalis (PPD) displayed gonadotrophic (GTH), thyrotrophic (TSH), somatotrophic (GH) and also a few PRL cells. Moreover, somatolactin (SL) and melanotrophic (MSH) cells were identified inside the pars intermedia (PI). Interestingly, some SL-immunoreactive fibers were also detected in the neurohypophysis. Some GTH cells were also located on the exterior surface of the PI. Glycoproteins containing mannose (Man) and/or glucose (Glc); N-acetyl-glucosamine (GlcNAc) and/or sialic acid sugar residues, as well as -S-S- groups, were observed in GTH, TSH and SL cells. The Bromophenol Blue technique stained amphiphilic SL, acidophilic GH cells and weakly ACTH cells. GH and ACTH cells were unreactive to PAS, Alcian Blue, Tioglycollate-Ferric-Ferricianide-FeIII and lectin (Con A and WGA) techniques. Finally, PAS reaction was positive in amphiphilic SL cells, which were PbH unreactive, while MSH and ACTH cells were stained with PbH technique.

Distribution of neuropeptide Y-like immunoreactivity in the brain of the Senegalese sole (Solea senegalensis).

We present the results of an immunohistochemical study aimed at localizing the neuropeptide Y (NPY) in the brain of the Senegalese sole, Solea senegalensis, using an antiserum raised against porcine NPY and the streptavidin-biotin-peroxidase method. In this species, we have identified immunoreactive cells in the ventral and dorsal telencephalon, caudal preoptic area, ventrocaudal hypothalamus, optic tectum, torus longitudinalis, synencephalon and isthmic region. NPY-immunoreactive fibers were profusely distributed throughout the brain, also reaching the adenohypophysis. The extensive distribution of NPY suggests an important role for this neuropeptide in a variety of physiological processes, including the neuroendocrine control of adenohypophyseal functions. Our results are compared with those obtained in other teleosts and discussed in relation to putative functions of NPY in the control of metabolism and reproduction in the Senegalese sole.

Cytoarchitectonic study of the brain of a perciform species, the sea bass (Dicentrarchus labrax). I. The telencephalon.

A cytoarchitectonic analysis of the telencephalon of the sea bass Dicentrarchus labrax, based on cresyl violet-stained serial transverse sections, is presented. Rostrally, the brain of the sea bass is occupied by sessile olfactory bulbs coupled to telencephalic hemispheres. The olfactory bulbs comprise an olfactory nerve fiber layer, a glomerular layer, an external cellular layer, a secondary olfactory fiber layer, and an internal cellular layer. Large terminal nerve ganglion cells are evident in the caudomedial olfactory bulbs. We recognized 22 distinct telencephalic nuclei which were classified in two main areas, the ventral telencephalon and the dorsal telencephalon. The ventral telencephalon displays four periventricular cell masses: the dorsal, ventral, supracommissural, and postcommissural nuclei; and four migrated populations: the lateral, central, intermediate, and entopeduncular nuclei. In addition, a periventricular cell population resembling the lateral septal organ reported in birds is observed in the ventral telencephalon of the sea bass. The dorsal telencephalon contains 13 nuclei, which can be organized into five major zones: the medial part, dorsal part, lateral part and its ventral, dorsal, and posterior divisions, the central part, and posterior part. Based on histological criteria, two cell masses are recognized in the ventral division of the lateral part of the dorsal telencephalon. The nucleus taenia is found in the caudal area of the dorsal telencephalon, close to the ventral area. This study represents a useful tool for the precise localization of the neuroendocrine territories and for the tracing of the neuronal systems participating in the regulation of reproduction and metabolism in this species.

Cytoarchitectonic study of the brain of a perciform species, the sea bass (Dicentrarchus labrax). II. The diencephalon.

The cytoarchitecture of nuclei in the preoptic area, ventral thalamus, dorsal thalamus, epithalamus, hypothalamus, posterior tuberculum, synencephalon, and pretectum and the accessory optic nuclei was analyzed in a perciform teleost, the sea bass Dicentrarchus labrax, by using serial sections stained with cresyl-violet. In general, the cytoarchitecture of the preoptic area, ventral and dorsal thalamus, epithalamus, and synencephalon resembles the histological pattern of other teleosts. However, the parvocellular preoptic nucleus of sea bass has been subdivided into parvocellular and anteroventral parts for morphological and functional reasons. The hypothalamus of the sea bass seems to differ slightly from that of other teleosts. An elaborated lateral tuberal nucleus, with five subdivisions, and three different nuclei around the lateral recesses were recognized. A medial nucleus of the inferior lobe, which has been reported previously in the perciform Sparus aurata, is also present in the hypothalamus of sea bass but has not been described before in another advanced teleost. The organization of the pretectum and the accessory optic system is essentially similar in sea bass to that described in other perciforms with highly developed vision. The migrated portion of the posterior tuberculum of sea bass appears to differ from this region of the diencephalon in other teleosts. In sea bass, three cell masses that have been described previously only in the perciform Sparus aurata have been assigned to the migrated area of the posterior tuberculum. This study will provide the neuroanatomical basis for future morpho-functional studies to be done in the sea bass brain.