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).

Target organs for lymphocystis disease virus replication in gilthead seabream (Sparus aurata).

The lymphocystis disease (LCD), the main viral pathology described in cultured gilthead seabream (Sparus aurata), is a self-limiting condition characterized by the appearance of hypertrophied fibroblasts (named lymphocysts) in the connective tissue of fish, primarily in the skin and fins. The causative agent of the disease is the Lymphocystis disease virus (LCDV), a member of the Iridoviridae family. In the present study, LCDV genome and transcripts were detected by real-time PCR in caudal fin, as well as in several internal organs, such as intestine, liver, spleen, kidney and brain, from asymptomatic, diseased and recovered gilthead seabream juveniles. These results indicate that the LCDV has a broad range tissue tropism, and can establish a systemic infection, even in subclinically infected fish. As showed by in situ hybridization, the permissive cells for LCDV infection seem to be fibroblasts, hepatocytes and cells of the mononuclear phagocyte system. Histopathological alterations associated with LCD were observed in all the organs analysed, including necrotic changes in liver and kidney, inflammatory response in the intestine submucosa or brain haemorrhage, although lymphocysts were only detected in the dermis of the caudal fin. Nevertheless, these histological changes were reverted in recovered animals.

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.

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.

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.

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.

Effects of the isoflavone daidzein in Senegalese sole, Solea senegalensis: Modulation of the oestrogen receptor-ß, apoptosis and enzymatic signalling pathways.

Phytochemicals are widely present in the aquatic environment and they are derived from many anthropogenic activities. The isoflavone daidzein is a natural compound that is found in the soya products used as habitual constituents of aquafeeds. Nevertheless, this isoflavone possesses oestrogenic and apoptotic properties. The present study determined the effects of daidzein (at 20 mg/L) during the first month and a half of life (from 7 to 44 days post-hatching -dph-) of the flatfish Senegalese sole, Solea senegalensis, focusing at the metamorphosis. We have analysed different gene expression levels and immunohistochemical protein patterns implicated in some oestrogenic, apoptosis and enzymatic pathways. In general, the oestrogen receptor (ERß) and stimulating apoptosis death receptor factor (Fas) transcript levels showed similar baseline patterns and transcriptional responses induced by daidzein. Both ERß and Fas were up-regulated by this isoflavone at the pre-metamorphosis and metamorphosis, and they were down-regulated in post-metamorphosed stages. The expression pattern of the apoptotic effector caspase (Casp6) was exclusively up-regulated at the pre-metamorphic phase. The Birc5 transcripts (i.e. anti-apoptosis, Survivin) were down-regulated by daidzein during certain metamorphic and post-metamorphosed stages. Besides, daidzein showed an up-regulating effect on both enzymatic complexes, the haemoprotein CYP1A and the acetylcholinesterase (AChE), except for a temporary AChE down-regulation in some post-metamorphosed stages. Immunostaining analysis only showed increased CYP1A signals in the liver of daidzein exposed fish. Overall, a majority of the transcriptional oestrogenic and apoptotic imbalances could be gradually and/or temporarily stabilised. Most controls and exposed larvae (70-80%) developed and grew following normal ontogenetic developmental patterns.

Toxicity and non-harmful effects of the soya isoflavones, genistein and daidzein, in embryos of the zebrafish, Danio rerio.

Based on the assumed oestrogenic and apoptotic properties of soya isoflavones (genistein, daidzein), and following the current OECD test-guidelines and principle of 3Rs, we have studied the potential toxicity of phytochemicals on the zebrafish embryos test (ZFET). For this purpose, zebrafish embryos at 2-3 h post-fertilisation (hpf) were exposed to both soya isoflavones (from 1.25 mg/L to 20 mg/L) and assayed until 96 hpf. Lethal and sub-lethal endpoints (mortality, hatching rates and malformations) were estimated in the ZFET, which was expanded to potential gene expression markers, determining the lowest observed effect (and transcriptional) concentrations (LOEC, LOTEC), and the no-observable effect (and transcriptional) concentrations (NOEC, NOTEC). The results revealed that genistein is more toxic (LC50-96 hpf: 4.41 mg/L) than daidzein (over 65.15 mg/L). Both isoflavones up-regulated the oestrogen (esrrb) and death receptors (fas) and cyp1a transcript levels. Most thyroid transcript signals were up-regulated by genistein (except for thyroid peroxidase/tpo), and the hatching enzyme (he1a1) was exclusively up-regulated by daidzein (from 1.25 mg/L onwards). The ZFET proved suitable for assessing toxicant effects of both isoflavones and potential disruptions (i.e. oestrogenic, apoptotic, thyroid, enzymatic) during the embryogenesis and the endotrophic larval period.

Toxicity of malathion at early life stages of the Senegalese sole, Solea senegalensis (Kaup, 1858): notochord and somatic disruptions.

The toxicity of malathion to Solea senegalensis was studied in a static renewal bioassay for 24, 48 and 72 h, with toxicant concentrations ranging from 1.56 until 100 µgL⁻¹. The LC50 values of malathion for 48 and 72 h was 63.5 (95% C.I: 50.83-79.34) and 22.94 (95% C.I: 17.16-30.68) µgL⁻¹ respectively. The survival of larvae was non-affected by exposure to malathion at concentrations up to 25 µgL⁻¹ (24 h NOEC), 6.25 µgL⁻¹ (48 h NOEC) and <1.6 µg⁻¹ (72 h NOEC). At the end of the experiment, surviving larvae from concentrations smaller than the 72h-LC50 were chosen to study morphological changes during malathion exposure. Results revealed a strong disruption in the notochord and trunk musculature integrity as a result of toxicant exposure. Noticeable changes in the composition and reduction of collagen fibers from the perinotochordal connective sheath and perimysium were clearly detected. The trunk musculature was also altered, showing a general disorganization of fibers. Moreover, malathion exposure provoked pericardial and yolk-sac oedemas and histopathological alterations in some other organ- systems and tissues (i.e. liver, pancreas, intestine).

Vitamin A Affects Flatfish Development in a Thyroid Hormone Signaling and Metamorphic Stage Dependent Manner.

Vitamin A (VA) and retinoid derivatives are known morphogens controlling vertebrate development. Despite the research effort conducted during the last decade, the precise mechanism of how VA induces post-natal bone changes, and particularly those operating through crosstalk with the thyroid hormones (THs) remain to be fully understood. Since effects and mechanisms seem to be dose and time-dependent, flatfish are an interesting study model as they undergo a characteristic process of metamorphosis driven by THs that can be followed by external appearance. Here, we studied the effects of VA imbalance that might determine Senegalese sole (Solea senegalensis) skeletogenetic phenotype through development of thyroid follicles, THs homeostasis and signaling when a dietary VA excess was specifically provided during pre-, pro- or post-metamorphic stages using enriched rotifers and Artemia as carriers. The increased VA content in enriched live prey was associated to a higher VA content in fish at all developmental stages. Dietary VA content clearly affected thyroid follicle development, T3 and T4 immunoreactive staining, skeletogenesis and mineralization in a dose and time-dependent fashion. Gene expression analysis showed that VA levels modified the mRNA abundance of VA- and TH-specific nuclear receptors at specific developmental stages. Present results provide new and key knowledge to better understand how VA and TH pathways interact at tissue, cellular and nuclear level at different developmental periods in Senegalese sole, unveiling how dietary modulation might determine juvenile phenotype and physiology.

Expression profiling of the sex-related gene Dmrt1 in adults of the Lusitanian toadfish Halobatrachus didactylus (Bloch and Schneider, 1801).

Doublesex and mab-3 related transcription factor 1 (Dmrt1) gene is a widely conserved gene involved in sex determination and differentiation across phyla. To gain insights on Dmrt1 implication for fish gonad cell differentiation and gametogenesis development, its mRNA was isolated from testis and ovary from the Lusitanian toadfish (Halobatrachus didactylus). The cDNA from Dmrt1 was synthesized and cloned, whereas its quantitative and qualitative gene expression, as well as its protein immunolocalization, were analyzed. A main product of 1.38 kb, which encodes a protein of 295 aa, was reported, but other minority Dmrt1 products were also identified by RACE-PCR. This gene is predominantly expressed in testis (about 20 times more than in other organs or tissues), specially in spermatogonia, spermatocytes and spermatids, as well as in somatic Sertoli cells, indicating that Dmrt1 plays an important role in spermatogenesis. Although Dmrt1 transcripts also seem to be involved in oogenesis development, and it cannot be excluded that toadfish Dmrt1 could be functionally involved in other processes not related to sex.