Characterisation of ChE in Solea solea and exposure of isoflavones in juveniles of commercial flatfish (Solea solea and Solea senegalensis).

The isoflavones genistein and daidzein are flavonoid compounds mainly found in legumes, especially in soybeans and their derived products. These flavonoids can be present in agricultural, domestic and industrial wastewater effluents as a result of anthropogenic activities and may be discharged in the environment. Due to the large growth of the aquaculture sector in recent decades, new and cost-effective fish feeds are being sought, but there is also a particular need to determine the effects of exposed flavonoids on fish in the aquatic environment, as this is the main route of exposure of organisms to endocrine disruptors. This study evaluated the possible effects of these isoflavones on juveniles of Solea senegalensis and Solea solea. After 48-96 h of exposure, the acetylcholinesterase activity in the sole head tissues was measured, and the cholinesterase activity in juveniles of common sole (S. solea) was determined. Experiments were carried out to determine the optimal pH, investigate the specificity of three substrates (acetylthiocholine, butyrylthiocholine, propionylthiocholine) on cholinesterase activity and determine the kinetic parameters (Vmax and Km ). The results obtained showed that neither genistein nor daidzein exposure to S. senegalensis and S. solea inhibited the activity of acetylcholinesterase at the tested concentrations (genistein: 1.25, 2.5, 5, 10 and 20 mg/L; daidzein: 0.625, 1.25, 2.5, 5 and 10 mg/L).

Post-thaw quality assessment of testicular fragments as a source of spermatogonial cells for surrogate production in the flatfish Solea senegalensis.

Cryopreservation of germ cells would facilitate the availability of cells at any time allowing the selection of donors and maintaining quality control for further applications such as transplantation and germline recovery. In the present study, we analyzed the efficiency of four cryopreservation protocols applied either to isolated cell suspensions or to testes fragments from Senegalese sole. In testes fragments, the quality of cryopreserved germ cells was analyzed in vitro in terms of cell recovery, integrity and viability, DNA integrity (fragmentation and apoptosis), and lipid peroxidation (malondialdehyde levels). Transplantation of cryopreserved germ cells was performed to check the capacity of cells to in vivo incorporate into the gonadal primordium of Senegalese sole early larval stages (6 days after hatching (dah), pelagic live), during metamorphosis (10 dah) and at post-metamorphic stages (16 dah and 20 dah, benthonic life). Protocols incorporating dimethyl sulfoxide (DMSO) as a cryoprotectant showed higher number of recovered spermatogonia, especially in samples cryopreserved with L-15 + DMSO (0.39 ± 0.18 × 106 cells). Lipid peroxidation and DNA fragmentation were also significantly lower in this treatment compared with other treatments. An important increase in oxidation (MDA levels) was detected in samples containing glycerol as a cryoprotectant, reflected also in terms of DNA damage. Transplantation of L-15 + DMSO cryopreserved germ cells into larvae during early metamorphosis (10 dah, 5.2 mm) showed higher incorporation of cells (27.30 ± 5.27%) than other larval stages (lower than 11%). Cryopreservation of germ cells using testes fragments frozen with L-15 + DMSO was demonstrated to be a useful technique to store Senegalese sole germline.

Toxicity of malathion during Senegalese sole, Solea senegalensis larval development and metamorphosis: Histopathological disorders and effects on type B esterases and CYP1A enzymatic systems.

The toxicity of malathion to Solea senegalensis was studied in a static renewal bioassay during its first month of larval life (between 4 and 30 dph). Through the use of different biomarkers and biochemical, cellular and molecular approaches (inhibition of cholinesterases [ChEs], changes in cytochrome P450-1A [CYP1A] and the study of histopathological alterations), the effects of three concentrations of malathion (1.56, 3.12, and 6.25 µg/L) have been analyzed. In subacute exposure, malathion inhibited cholinesterase activities (AChE, BChE, CbE) in a dose- and time-dependent manner, ranging the inhibition percentage from 20% to 90%. However, the expression levels of CYP1A and AChE transcripts or proteins were not modified. Additionally, exposure to malathion provoked histopathological alterations in several organ systems of Senegalese sole in a time- and dose dependent way, namely disruption of parenchymal architecture in the liver, epithelial desquamation, pyknotic nuclei and steatosis in the intestine, disorganization of supporting cartilage, and sings of hyperplasia and hypertrophy in the gills and degeneration of the epithelial cells from the renal tubules. Malathion exposure also provoked strong disorganization of cardiac fibers from the heart. The findings provide evidence that exposure to sublethal concentrations of malathion that provoked serious injury to the fish S. senegalensis, were below the expected environmental concentrations reported in many other ecosystems and different fish species,revealing a higher sensitivity for Solea senegalensis to malathion exposure, thus reinforcing its use as sentinel species for environmental pollution in coastal and estuarine environments.

Feed and immersion challenges with lymphocystis disease virus (LCDV) reveals specific mechanisms for horizontal transmission and immune response in senegalese sole post-larvae.

The horizontal transmission of lymphocystis disease virus (LCDV) through contaminated water and feed (using artemia as vehicle) and the associated immune gene expression profiles in Senegalese sole post-larvae were investigated. All specimens analyzed were positive for LCDV DNA detection at 1-day post-challenge (1 dpc) with the highest viral levels in specimens infected through the immersion route. However, the percentage of LCDV-positive animals and number of viral DNA copies dropped progressively at 2 and 7 dpc. The histological analysis identified structural changes in the skin, muscle and gills of sole post-larvae LCDV-challenged by immersion. In situ hybridization confirmed a wide distribution of LCDV in the skin, gut, surrounding vessels in trunk muscle and head kidney in the immersion route, while the signals were restricted to the liver and lamina propria in the feeding treatment. Expression analysis using a set of 22 genes related to innate immune defense system demonstrated clear differences in the time-course response to LCDV as function of the infection route. Most antiviral defense genes, the proinflammatory cytokines, the complement c3, g-type lysozyme and T-cell markers cd4 and cd8a were rapidly induced in the feeding-infected post-larvae, and they were remained activated at 2 dpc. In contrast, in the immersion-infected post-larvae the induction of most defensive genes was delayed, with a low intensity at 2 dpc. All these data demonstrate that LCDV can horizontally infect Senegalese sole post-larvae through the water or feed although with different patterns of histopathological disorders, virus distribution and route-specific expression profiles.

The organophosphate pesticide -OP- malathion inducing thyroidal disruptions and failures in the metamorphosis of the Senegalese sole, Solea senegalensis.

BACKGROUND: Organophosphate pesticides-OP-, like malathion, can alter the normal functioning of neuro-endocrine systems (e.g., hypothalamus-pituitary-thyroid-HPT- axis), and to interfere on the thyroidal homeostasis. Through direct interactions with thyroid receptors, an/or indirectly via up-stream signalling pathways, from the HPT axis (i.e., negative feedback regulation), malathion possess the ability to affect integrity of thyroidal follicular tissue, and it can also block or delay its hormonal functioning. This insecticide can alter the majority of the ontogenetic processes, inducing several deformities, and also provoking decreases in the growth and survival patterns. The present study has been performed to determine the sublethal effects of malathion during the first month of life of the Senegalese sole, Solea senegalensis, and it is mainly focused on the metamorphosis phase. Different transcript expression levels (i.e. thyroid receptors, matrix and bone -Gla-proteins) and immunohistochemical patterns (i.e. thyroid hormones, osteocalcin, cell proliferation) have been analysed during the most critical phases of the flatfish metamorphosis, that is, through differentiation of thyroid system and skeletal development, migration of the eye, and further adaptation to benthic behaviours. RESULTS: In early life stages of the Senegalese sole, the exposure to the highest concentration of malathion (6.25 µg/L) affected to the growth patterns, showing the exposed individuals, a reduction around 60 and 92% of the total length and the dry weigth, respectively. In paralell, a significant reduction of the thyroid follicles (i.e., size and number) it was also been recorded, in a dose-dependent way. Abnormal phenotypes induced in the exposed larvae, did not complete the process of metamorphosis, and displayed several morphological abnormalities and developmental disorders, which were mainly associated with the eye migration process, and with thyroidal and skeletal disorders (i.e., transcriptional and protein changes of thyroid hormones and receptors, and of matrix and bone Gla proteins distribution), that conduced to an inadequate adaptation to the benthic life. CONCLUSIONS: In the Senegalese sole, the majority of the ontogenetic alterations induced by the exposure to malathion were mainly associated to the metamorphosis period, which is a thyroid-driven proccess. In fact, most crucial and transitional ontogenic events, appeared notably disturbed, for e.g., thyroid gland differentiation and functioning, migration of eye, skeletal development and benthonic behaviors.

Warfarin-exposed zebrafish embryos resembles human warfarin embryopathy in a dose and developmental-time dependent manner - From molecular mechanisms to environmental concerns.

Warfarin is the most worldwide used anticoagulant drug and rodenticide. Since it crosses placental barrier it can induce warfarin embryopathy (WE), a fetal mortality in neonates characterized by skeletal deformities in addition to brain hemorrhages. Although the effects of warfarin exposure in aquatic off target species were already described, the particular molecular toxicological mechanisms during early development are still unclear. Here, we used zebrafish (Danio rerio) to describe and compare the developmental effects of warfarin exposure (0, 15.13, 75.68 and 378.43 mM) on two distinct early developmental phases (embryos and eleuthero-embryos). Although exposure to both developmental phases induced fish mortality, only embryos exposed to the highest warfarin level exhibited features mimicking mammalian WE, e.g. high mortality, higher incidence of hemorrhages and altered skeletal development, among other effects. To gain insights into the toxic mechanisms underlying warfarin exposure, the transcriptome of embryos exposed to warfarin was explored through RNA-Seq and compared to that of control embryos. 766 differentially expressed (564 up- and 202 down-regulated) genes were identified. Gene Ontology analysis revealed particular cellular components (cytoplasm, extracellular matrix, lysosome and vacuole), biological processes (mainly amino acid and lipid metabolism and response to stimulus) and pathways (oxidative stress response and apoptosis signaling pathways) being significantly overrepresented in zebrafish embryos upon warfarin exposure. Protein-protein interaction further evidenced an altered redox system, blood coagulation and vasculogenesis, visual phototransduction and collagen formation upon warfarin exposure. The present study not only describes for the first time the WE in zebrafish, it provides new insights for a better risk assessment, and highlights the need for programming the rat eradication actions outside the fish spawning season to avoid an impact on off target fish community. The urge for the development of more species-specific anticoagulants for rodent pest control is also highlighted.

Effects of the isoflavone genistein in early life stages of the Senegalese sole, Solea senegalensis: role of the Survivin and proliferation versus apoptosis pathways.

BACKGROUND: Phytochemical flavonoids are widely distributed in the environment and are derived from many anthropogenic activities. The isoflavone genistein is a naturally occurring compound found in soya products that are habitual constituents of the aquafeeds. This isoflavone possesses oestrogenic biological activity and also apoptotic properties. The present study has been performed to determine the effects of the genistein in the early life stages of the flatfish Senegalese sole during the first month of larval life, and it is focused especially at the metamorphosis, analysing the expression transcript levels and the immunohistochemical protein patterns implicated in the cell proliferation and apoptosis pathways (proliferation cellular/PCNA, anti-apoptosis Survivin/BIRC-5, death receptors/Fas, and Caspases). RESULTS: The isoflavone genistein induced some temporal disrupting effects in several pro-apoptotic signalling pathways (Fas, CASP-6) at both genistein doses (3 mg/L and 10 mg/L), with increased Fas transcripts and also decreasing CASP-6 mRNA expression levels during metamorphic and post-metamorphic stages of the Senegalese sole. On the other hand, the anti-apoptotic BIRC-5 expression levels were weakly down-regulated with both the highest and lowest doses, but all of these imbalances were stabilised to the baseline levels. In early life stages of the controls, the constitutive basal transcript levels were temporarily and differentially expressed, reaching the highest levels at the pre-metamorphosis phase, as especially in endotrophic larvae (i.e. BIRC-5 mRNA), as well as in the metamorphic (i.e. CASP-6 mRNA) and post-metamorphic stages (i.e. Fas mRNA). In general, through development, continuous and progressive increases in the protein patterns of cell proliferation-PCNA (e.g. mitotic nuclei), anti-apoptotic Survivin (e.g. haematopoietic system, brain, digestive system, gills) and CASP-2 and -6 (e.g. brain, gills, kidney, digestive system, vascular systems, among others) have been immunohistochemically detected. Besides, both the controls and genistein exposed larvae displayed parallel immunostaining protein patterns in the different organ-systems and tissues. CONCLUSIONS: The transcriptional imbalances observed in the studied genes (BIRC-5, CASP-6, Fas) were only temporarily induced, and apparently no changes in the immunohistochemical protein patterns were detected. Thus, the isoflavone genistein caused not harmful effects in the development and metamorphosis of the Senegalese sole exposed to chronic environmentally relevant concentrations (3 and 10 mg/L).

Effects of the soya isoflavone genistein in early life stages of the Senegalese sole, Solea senegalensis: Thyroid, estrogenic and metabolic biomarkers.

This study examines the effects induced by environmentally relevant concentrations of the isoflavone genistein (3mg/L and 10mg/L) during early life stages of the Senegalese sole. Throughout the hypothalamus-pituitary-thyroid (HPT) axis, several neurohormonal regulatory thyroid signalling patterns (thyroglobulin/Tg, thyroid peroxidase/TPO, transthyretin/TTR, thyroid receptors/TRß, and iodothrynonine deiodinases, Dio2 and Dio3) were analysed. Furthermore, the expression patterns of estrogen receptor ERß and haemoprotein Cyp1a were also evaluated. In the control larvae, progressive increases of constitutive hormonal signalling pathways have been evidenced from the pre-metamorphosis phase onwards, reaching the highest expression basal levels at the metamorphosis (Tg, TPO, Dio2) and/or during post-metamorphosis (TTR, TRß, ERß). When the early larvae were exposed to both genistein concentrations (3mg/L and 10mg/L), a statistically significant down-regulation of TPO, TTR and Tg mRNA levels was clearly detected at the metamorphic stages. In addition, the Dio2 and Dio3 transcript expression levels were also down and up-regulated when exposed to both genistein concentrations. In the larvae exposed to genistein, no statistically significant responses were recorded for the TRß expression patterns. Nevertheless, the ERß and Cyp1a transcript levels were up-regulated at the middle metamorphic stage (S2, at 16 dph) in the larvae exposed to high genistein concentrations and, only the ERß was down-regulated (S1, at 12dph) at the lower doses. Finally, all these pointed out imbalances were only temporarily disrupted by exposure to genistein, since most of the modulated transcriptional signals (i.e. up or down-regulation) were quickly restored to the baseline levels. Additionally, the control and genistein-exposed Senegalese sole specimens showed characteristic ontogenetic patterns and completely suitable for an optimal development, metamorphosis, and growth.

Development of interspecies testicular germ-cell transplantation in flatfish.

Interspecific testicular germ cell (TGC) transplantation was investigated in two commercial flatfish species. Testes from donor species (Senegalese sole) were evaluated using classical histological techniques (haematoxylin-eosin staining and haematoxylin-light green-orange G-acid fuchsine staining), in situ hybridisation and immunohistochemical analysis. Both Ssvasa1-2 mRNAs and SsVasa protein allowed the characterisation of TGCs, confirming the usefulness of the vasa gene in the detection of Senegalese sole TGCs. Xenogenic transplants were carried out using TGCs from one-year-old Senegalese sole into turbot larvae. Propidium iodide-SYBR-14 and 4',6'-diamidino-2-phenylindole (DAPI) staining showed that 87.98% of the extracted testicular cells were viable for microinjection and that 15.63% of the total recovered cells were spermatogonia. The vasa gene was characterised in turbot recipients using cDNA cloning. Smvasa mRNA was confirmed as a germ cell-specific molecular marker in this species. Smvasa expression analysis during turbot ontogeny was carried out before Senegalese sole TGC transplants into turbot larvae. Turbot larvae at 18 days after hatching (DAH) proved to be susceptible to manipulation procedures. High survival rates (83.75±15.90-100%) were obtained for turbot larvae at 27, 34 and 42 DAH. These data highlight the huge potential of this species for transplantation studies. Quantitative PCR was employed to detect Senegalese sole vasa mRNAs (Ssvasa1-2) in the recipient turbot larvae. The Ssvasa mRNAs showed a significant increase in relative expression in 42-DAH microinjected larvae three weeks after treatment, showing the proliferation of Senegalese sole spermatogonia in transplanted turbot larvae.

Solea senegalensis vasa transcripts: molecular characterisation, tissue distribution and developmental expression profiles.

The Vasa protein is an RNA helicase belonging the DEAD (Asp-Glu-Ala-Asp)-box family. The crucial role played by the vasa gene in the germ-cell lineage of both vertebrates and invertebrates has made this gene a useful molecular marker for germinal cells and a useful tool in surrogate broodstock production using primordial germ cell transplantation. With the aim of establishing a novel approach to improving Solea senegalensis broodstock management, the vasa gene in this species was characterised. Four S. senegalensis vasa transcripts were isolated: Ssvasa1, Ssvasa2, Ssvasa3 and Ssvasa4. Their phylogenetic relationship with other vasa homologues was determined confirming the high degree of conservation of this helicase throughout evolution. Our qPCR results showed that S. senegalensis vasa transcripts are prevalently expressed in gonads, with ovary-specific expression for Ssvasa3 and Ssvasa4. During embryonic and larval development, a switch between the longest and the shortest transcripts was observed. While Ssvasa1 and Ssvasa2 were maternally supplied, Ssvasa3 and Ssvasa4 depended on the de novo expression program of the growing juveniles, suggesting that vasa mRNA could be involved in Senegalese sole gonad differentiation. In situ hybridisation and immunohistochemical analysis performed in 150-days after hatching (DAH) larvae showed vasa product expression in the germinal region of early gonads. In our work we demonstrated the usefulness of Ssvasa mRNAs as molecular markers for primordial germ cells and germinal cells during embryonic development, larval ontogenesis and gonad differentiation. Furthermore, our results confirmed the potential of vasa to help investigate germinal cell biotechnology for Senegalese sole reproduction.

Specific Blood Plasma Circulating miRs Are Associated with the Physiological Impact of Total Fish Meal Replacement with Soybean Meal in Diets for Rainbow Trout (Oncorhynchus mykiss).

High dietary SBM content is known to induce important physiological alterations, hampering its use as a major FM alternative. Rainbow trout (Oncorhynchus mykiss) juveniles were fed two experimental diets during 9 weeks: (i) a FM diet containing 12% FM; and (ii) a vegetable meal (VM) diet totally devoid of FM and based on SBM (26%). Fish fed the VM diet did not show reduced growth performance when compared with fish fed the FM diet. Nevertheless, fish fed the VM diet had an increased viscerosomatic index, lower apparent fat digestibility, higher aminopeptidase enzyme activity and number of villi fusions, and lower α-amylase enzyme activity and brush border integrity. Small RNA-Seq analysis identified six miRs (omy-miR-730a-5p, omy-miR-135c-5p, omy-miR-93a-3p, omy-miR-152-5p, omy-miR-133a-5p, and omy-miR-196a-3p) with higher expression in blood plasma from fish fed the VM diet. Bioinformatic prediction of target mRNAs identified several overrepresented biological processes known to be associated with high dietary SBM content (e.g., lipid metabolism, epithelial integrity disruption, and bile acid status). The present research work increases our understanding of how SBM dietary content has a physiological impact in farmed fish and suggests circulating miRs might be suitable, integrative, and less invasive biomarkers in fish.

Debaryomyces hansenii supplementation in low fish meal diets promotes growth, modulates microbiota and enhances intestinal condition in juvenile marine fish.

BACKGROUND: The development of a sustainable business model with social acceptance, makes necessary to develop new strategies to guarantee the growth, health, and well-being of farmed animals. Debaryomyces hansenii is a yeast species that can be used as a probiotic in aquaculture due to its capacity to i) promote cell proliferation and differentiation, ii) have immunostimulatory effects, iii) modulate gut microbiota, and/or iv) enhance the digestive function. To provide inside into the effects of D. hansenii on juveniles of gilthead seabream (Sparus aurata) condition, we integrated the evaluation of the main key performance indicators coupled with the integrative analysis of the intestine condition, through histological and microbiota state, and its transcriptomic profiling. RESULTS: After 70 days of a nutritional trial in which a diet with low levels of fishmeal (7%) was supplemented with 1.1% of D. hansenii (17.2 × 105 CFU), an increase of ca. 12% in somatic growth was observed together with an improvement in feed conversion in fish fed a yeast-supplemented diet. In terms of intestinal condition, this probiotic modulated gut microbiota without affecting the intestine cell organization, whereas an increase in the staining intensity of mucins rich in carboxylated and weakly sulphated glycoconjugates coupled with changes in the affinity for certain lectins were noted in goblet cells. Changes in microbiota were characterized by the reduction in abundance of several groups of Proteobacteria, especially those characterized as opportunistic groups. The microarrays-based transcriptomic analysis found 232 differential expressed genes in the anterior-mid intestine of S. aurata, that were mostly related to metabolic, antioxidant, immune, and symbiotic processes. CONCLUSIONS: Dietary administration of D. hansenii enhanced somatic growth and improved feed efficiency parameters, results that were coupled to an improvement of intestinal condition as histochemical and transcriptomic tools indicated. This probiotic yeast stimulated host-microbiota interactions without altering the intestinal cell organization nor generating dysbiosis, which demonstrated its safety as a feed additive. At the transcriptomic level, D. hansenii promoted metabolic pathways, mainly protein-related, sphingolipid, and thymidylate pathways, in addition to enhance antioxidant-related intestinal mechanisms, and to regulate sentinel immune processes, potentiating the defensive capacity meanwhile maintaining the homeostatic status of the intestine.

Evidence for 5S rDNA horizontal transfer in the toadfish Halobatrachus didactylus (Schneider, 1801) based on the analysis of three multigene families.

BACKGROUND: The Batrachoididae family is a group of marine teleosts that includes several species with more complicated physiological characteristics, such as their excretory, reproductive, cardiovascular and respiratory systems. Previous studies of the 5S rDNA gene family carried out in four species from the Western Atlantic showed two types of this gene in two species but only one in the other two, under processes of concerted evolution and birth-and-death evolution with purifying selection. Here we present results of the 5S rDNA and another two gene families in Halobatrachus didactylus, an Eastern Atlantic species, and draw evolutionary inferences regarding the gene families. In addition we have also mapped the genes on the chromosomes by two-colour fluorescence in situ hybridization (FISH). RESULTS: Two types of 5S rDNA were observed, named type α and type ß. Molecular analysis of the 5S rDNA indicates that H. didactylus does not share the non-transcribed spacer (NTS) sequences with four other species of the family; therefore, it must have evolved in isolation. Amplification with the type ß specific primers amplified a specific band in 9 specimens of H. didactylus and two of Sparus aurata. Both types showed regulatory regions and a secondary structure which mark them as functional genes. However, the U2 snRNA gene and the ITS-1 sequence showed one electrophoretic band and with one type of sequence. The U2 snRNA sequence was the most variable of the three multigene families studied. Results from two-colour FISH showed no co-localization of the gene coding from three multigene families and provided the first map of the chromosomes of the species. CONCLUSIONS: A highly significant finding was observed in the analysis of the 5S rDNA, since two such distant species as H. didactylus and Sparus aurata share a 5S rDNA type. This 5S rDNA type has been detected in other species belonging to the Batrachoidiformes and Perciformes orders, but not in the Pleuronectiformes and Clupeiformes orders. Two hypotheses have been outlined: one is the possible vertical permanence of the shared type in some fish lineages, and the other is the possibility of a horizontal transference event between ancient species of the Perciformes and Batrachoidiformes orders. This finding opens a new perspective in fish evolution and in the knowledge of the dynamism of the 5S rDNA. Cytogenetic analysis allowed some evolutionary trends to be roughed out, such as the progressive change in the U2 snDNA and the organization of (GATA)n repeats, from dispersed to localized in one locus. The accumulation of (GATA)n repeats in one chromosome pair could be implicated in the evolution of a pair of proto-sex chromosomes. This possibility could situate H. didactylus as the most highly evolved of the Batrachoididae family in terms of sex chromosome biology.

Short-term exposure to pharmaceuticals negatively impacts marine flatfish species: Histological, biochemical and molecular clues for an integrated ecosystem risk assessment.

The marine habitat and its biodiversity can be impacted by released pharmaceuticals. The short-term (7 days) effect of 3 commonly used drugs - warfarin, dexamethasone and imidazole - on Senegalese sole (Solea senegalensis) juveniles was investigated. Occurrence of hemorrhages, histopathological alterations, antioxidant status, activity of antioxidant enzymes and expression of genes involved in the xenobiotic response (pxr, abcb1 and cyp1a), were evaluated. The results showed a time and drug-dependent effect. Warfarin exposure induced hemorrhages, hepatocyte vacuolar degeneration, and altered the activity of glutathione peroxidase (GPx) and the expression of all the studied genes. Dexamethasone exposure increased liver glycogen content, altered antioxidant status, GPx and superoxide dismutase activities, as well as abcb1 and cyp1a expression. Imidazole induced hepatocyte vacuolar degeneration and ballooning, and altered the antioxidant status and expression of the tested genes. The present work anticipates a deeper impact of pharmaceuticals on the aquatic environment than previously reported, thus underlining the urgent need for an integrated risk assessment.

Effects of in vivo treatment with the dopamine antagonist pimozide and gonadotropin-releasing hormone agonist (GnRHa) on the reproductive axis of Senegalese sole (Solea senegalensis).

The Senegalese sole (Solea senegalensis) is a flatfish that exhibits severe reproductive dysfunctions in captivity. This study aimed at investigating the existence of a dopamine (DA) inhibitory tone on the reproductive axis of this species. Four groups of Senegalese sole breeders were treated with, saline (controls, CNT), the DA antagonist pimozide (PIM, 5 mg kg(-1)), gonadotropin-releasing hormone agonist (GnRHa, 40 µg kg(-1)) or a combination of PIM+GnRHa (COMB). Effects were evaluated on pituitary GnRH levels (ELISA), pituitary gonadotropin subunit transcript levels (qPCR), plasma levels of sex steroids and vitellogenin (ELISA), gonad development (histology), spermiation and egg production. The GnRHa treatment induced egg release and stimulated testis maturation. In males, PIM did not affect pituitary GnRH content, but enhanced GnRHa-induced pituitary GPα transcripts and modified plasma androgen levels; moreover, PIM stimulated spermatogenesis and milt production, both alone and combined with GnRHa. In females, PIM did not affect pituitary and plasma endocrine parameters and did not affect egg production and fertilization success of the broodstock, either alone or in the combined treatment. In conclusion, data indicated the existence of a DA inhibition in mature males, which would be absent or weakly expressed in females.

Medicinal Plant Leaf Extract From Sage and Lemon Verbena Promotes Intestinal Immunity and Barrier Function in Gilthead Seabream (Sparus aurata).

The inclusion of a medicinal plant leaf extract (MPLE) from sage (Salvia officinalis) and lemon verbena (Lippia citriodora), rich in verbascoside and triterpenic compounds like ursolic acid, was evaluated in gilthead seabream (Sparus aurata) fed a low fishmeal-based diet (48% crude protein, 17% crude fat, 21.7 MJ kg-1, 7% fishmeal, 15% fish oil) for 92 days. In particular, the study focused on the effect of these phytogenic compounds on the gut condition by analyzing the transcriptomic profiling (microarray analysis) and histological structure of the intestinal mucosa, as well as the histochemical properties of mucins stored in goblet cells. A total number of 506 differentially expressed genes (285 up- and 221 down-regulated) were found when comparing the transcriptomic profiling of the intestine from fish fed the control and MPLE diets. The gut transcripteractome revealed an expression profile that favored biological mechanisms associated to the 1) immune system, particularly involving T cell activation and differentiation, 2) gut integrity (i.e., adherens and tight junctions) and cellular proliferation, and 3) cellular proteolytic pathways. The histological analysis showed that the MPLE dietary supplementation promoted an increase in the number of intestinal goblet cells and modified the composition of mucins' glycoproteins stored in goblet cells, with an increase in the staining intensity of neutral mucins, as well as in mucins rich in carboxylated and weakly sulfated glycoconjugates, particularly those rich in sialic acid residues. The integration of transcriptomic and histological results showed that the evaluated MPLE from sage and lemon verbena is responsible for the maintenance of intestinal health, supporting gut homeostasis and increasing the integrity of the intestinal epithelium, which suggests that this phytogenic may be considered as a promising sustainable functional additive for aquafeeds.

Chromosomal mapping of the major and minor ribosomal genes, (GATA)n and U2 snRNA gene by double-colour FISH in species of the Batrachoididae family.

In the present study dual-colour fluorescence in situ hybridization (FISH) was performed to study the chromosomal distribution of 18S and 5S rDNAs, (GATA)(n) and 5S rDNA, and U2 snRNA and 18S rDNA in four species of Batrachoididae family: Amphichthys cryptocentrus, Batrachoides manglae, Porichthys plectrodon and Thalassophryne maculosa. The 18S rDNA signals were present in only one pair of chromosomes in all the four Batrachoididae species. The 5S rDNA was mapped on one pair of chromosomes, except in B. manglae, which showed a hybridization signal in two pairs. The two ribosomal genes are located on different chromosome pairs, except in A. cryptocentrus, in which they appear co-located. In all the cases, the (GATA)(n) probe produced disperse hybridization signals in all four species. The U2 snRNA signals appear very widely scattered in A. cryptocentrus, P. plectrodon, but show a degree of clustering in a specific chromosome pair in B. manglae. In T. maculosa, they are thinly dispersed and strong hybridization signals are observed co-located to the 18S rDNA-bearing chromosomes. Finally, a double-colour FISH with U2 snRNA and 5S rDNA probes was performed in B. manglae, and this showed that these genes were not co-located. These results have been compared with those from another Batrachoididae species, and evolutive processes of these species are discussed.

Sequence characterization and phylogenetic analysis of the 5S ribosomal DNA in species of the family Batrachoididae.

5S ribosomal DNA (rDNA) sequences were analyzed in four species belonging to different genera of the fish family Batrachoididae. Several 5S rDNA variants differing in their non-transcribed spacers (NTSs) were found and were grouped into two main types. Two species showed both types of 5S rDNA, whereas the other two species showed only one type. One type of NTS of Amphichthys cryptocentrus showed a high polymorphism due to several deletions and insertions, and phylogenetic analysis showed a between-species clustering of this type of NTS in Amphichthys cryptocentrus. These results suggest a clear differentiation in the model of 5S rDNA evolution of these four species of Batrachoididae, which appear to have been subject to processes of concerted evolution and birth-and-death evolution with purifying selection.

Toxicity of parathion on embryo and yolk-sac larvae of gilthead seabream (Sparus aurata l.): effects on survival, cholinesterase, and carboxylesterase activity.

This study was conducted to examine the acute toxicity of the organophosphorus pesticide (OP) parathion on embryos and yolk-sac larvae of gilthead seabream (Sparus aurata), and to investigate the effects of this compound on cholinesterase and carboxylesterase activity of seabream larvae in the phase of endogenous feeding. The 72-h LC50 for yolk-sac larvae (0.523 mg L⁻¹) was about two-fold lower than the 48-h LC50 for embryos (1.005 mg L⁻¹). Parathion significantly inhibited the activity of ChE and CaE activity in yolk sac larvae but there were not significant differences in the sensitivity of both esterases to parathion as inferred by their 72-h IC50 values. Larvae exposed to parathion for 72 h showed a 70% inhibition of the whole body acetylcholinesterase at approximately the LC50.

Unveiling the effect of dietary essential oils supplementation in Sparus aurata gills and its efficiency against the infestation by Sparicotyle chrysophrii.

A microencapsulated feed additive composed by garlic, carvacrol and thymol essential oils (EOs) was evaluated regarding its protective effect in gills parasitized by Sparicotyle chrysophrii in Sparus aurata. A nutritional trial (65 days) followed by a cohabitation challenge with parasitized fish (39 days) were performed. Transcriptomic analysis by microarrays of gills of fish fed the EOs diet showed an up-regulation of genes related to biogenesis, vesicular transport and exocytosis, leukocyte-mediated immunity, oxidation-reduction and overall metabolism processes. The functional network obtained indicates a tissue-specific pro-inflammatory immune response arbitrated by degranulating acidophilic granulocytes, sustained by antioxidant and anti-inflammatory responses. The histochemical study of gills also showed an increase of carboxylate glycoproteins containing sialic acid in mucous and epithelial cells of fish fed the EOs diet, suggesting a mucosal defence mechanism through the modulation of mucin secretions. The outcomes of the in vivo challenge supported the transcriptomic results obtained from the nutritional trial, where a significant reduction of 78% in the abundance of S. chrysophrii total parasitation and a decrease in the prevalence of most parasitic developmental stages evaluated were observed in fish fed the EOs diet. These results suggest that the microencapsulation of garlic, carvacrol and thymol EOs could be considered an effective natural dietary strategy with antiparasitic properties against the ectoparasite S. chrysophrii.