Percorrer por autor "Manchado, Manuel"
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- A thyroid hormone regulated asymmetric responsive centre is correlated with eye migration during flatfish metamorphosisPublication . Campinho, Marco António; Silva, Nádia; Martins, Gabriel G.; Anjos, Liliana; Florindo, Claudia; Roman-Padilla, Javier; Garcia-Cegarra, Ana; Louro, Bruno; Manchado, Manuel; Power, DeborahFlatfish metamorphosis is a unique post-embryonic developmental event in which thyroid hormones (THs) drive the development of symmetric pelagic larva into asymmetric benthic juveniles. One of the eyes migrates to join the other eye on the opposite side of the head. Developmental mechanisms at the basis of the acquisition of flatfish anatomical asymmetry remain an open question. Here we demonstrate that an TH responsive asymmetric centre, determined by deiodinase 2 expression, ventrally juxtaposed to the migrating eye in sole (Solea senegalensis) correlates with asymmetric cranial ossification that in turn drives eye migration. Besides skin pigmentation that is asymmetric between dorsal and ventral sides, only the most anterior head region delimited by the eyes becomes asymmetric whereas the remainder of the head and organs therein stay symmetric. Sub-ocular ossification is common to all flatfish analysed to date, so we propose that this newly discovered mechanism is universal and is associated with eye migration in all flatfish.
- Deciphering the role of cartilage protein 1 in human dermal fibroblasts: a transcriptomic approachPublication . Letsiou, Sophia; Manchado, Manuel; Zografaki, Mariela; Marka, Sofia; L, Anjos; Skliros, Dimitrios; Martínez-Blanch, Juan F.; Flemetakis, E.; Power, DeborahCartilage acidic protein 1A (hCRTAC1-A) is an extracellular matrix protein (ECM) of human hard and soft tissue that is associated with matrix disorders. The central role of fibroblasts in tissue integrity and ECM health made primary human dermal fibroblasts (NHDF) the model for the present study, which aimed to provide new insight into the molecular function of hCRTAC1-A. Specifically, we explored the differential expression patterns of specific genes associated with the presence of hCRTAC1-A by RNA-seq and RT-qPCR analysis. Functional enrichment analysis demonstrated, for the very first time, that hCRTAC1-A is involved in extracellular matrix organization and development, through its regulatory effect on asporin, decorin, and complement activity, in cell proliferation, regeneration, wound healing, and collagen degradation. This work provides a better understanding of putative hCRTAC1-A actions in human fibroblasts and a fundamental insight into its function in tissue biology.
- Dietary antioxidant supplementation promotes growth in senegalese sole postlarvaePublication . Xavier, Maria J; Engrola, Sofia; Conceição, Luis E C; Manchado, Manuel; Carballo, Carlos; Gonçalves, Renata; Colen, R.; Figueiredo, Vera; Valente, Luisa M PSomatic growth is a balance between protein synthesis and degradation, and it is largely influenced by nutritional clues. Antioxidants levels play a key role in protein turnover by reducing the oxidative damage in the skeletal muscle, and hence promoting growth performance in the long-term. In the present study, Senegalese sole postlarvae (45 days after hatching, DAH) were fed with three experimental diets, a control (CTRL) and two supplemented with natural antioxidants: curcumin (CC) and grape seed (GS). Trial spanned for 25 days and growth performance, muscle cellularity and the expression of muscle growth related genes were assessed at the end of the experiment (70 DAH). The diets CC and GS significantly improved growth performance of fish compared to the CTRL diet. This enhanced growth was associated with larger muscle cross sectional area, with fish fed CC being significantly different from those fed the CTRL. Sole fed the CC diet had the highest number of muscle fibers, indicating that this diet promoted muscle hyperplastic growth. Although the mean fiber diameter did not differ significantly amongst treatments, the proportion of large-sized fibers (>25 μm) was also higher in fish fed the CC diet suggesting increased hypertrophic growth. Such differences in the phenotype were associated with a significant up-regulation of the myogenic differentiation 2 (myod2) and the myomaker (mymk) transcripts involved in myocyte differentiation and fusion, respectively, during larval development. The inclusion of grape seed extract (GS diet) resulted in a significant increase in the expression of myostatin1. These results demonstrate that both diets (CC and GS) can positively modulate muscle development and promote growth in sole postlarvae. This effect is more prominent in CC fed fish, where increased hyperplastic and hypertrophic growth of the muscle was associated with an upregulation of myod2 and mymk genes.
- Dietary natural plant extracts can promote growth and modulate oxidative status of Senegalese Sole Postlarvae under standard/challenge conditionsPublication . Xavier, Maria João; Conceição, Luís E. C.; Valente, Luisa M. P.; Colen, R.; Rodrigues, Andreia C. M.; Rocha, Rui J. M.; L, Custódio; Carballo, Carlos; Manchado, Manuel; Engrola, SofiaPlant extracts are known for their high content and diversity of polyphenols, which can improve fish oxidative status. A growth trial with Senegalese sole postlarvae (45 days after hatching) fed with one of four experimental diets—control (CTRL), and supplemented with curcumin (CC), green tea (GT), and grape seed (GS) extracts—was performed to assess if supplementation could improve growth performance and oxidative status. At the end of the growth trial, postlarvae were submitted to a thermal stress to assess their robustness. Sole growth was improved by CC and GS diets when compared to those fed the CTRL. CC and CTRL postlarvae presented the lowest oxidative damage (lipid peroxidation and protein carbonylation values). Stress-related biomarkers (heat shock protein 70 and glutathione-S-transferase) decreased in CC fish compared to those fed the CTRL diet, which might be due to a direct antioxidant capacity. In contrast, oxidative damage increased in GT and GS sole reared in standard conditions. However, after a thermal stress, GT and GS diets prevented the increase of protein carbonylation content and the decrease of antioxidant glutathione, depending on exposure time. Overall, dietary supplementation with natural extracts modulated oxidative status and stress response after a short/long-term exposure to temperature.
- Insights into core molecular changes associated with metamorphosis in gilthead seabream larvae across diverse hatcheriesPublication . NAJAFPOUR, BABAK; Santos, Soraia; Manchado, Manuel; Vidal, Aurora; Tsipourlianos, Andreas; Canario, Adelino; Moutou, Katerina A.; Power, Deborah MaryEarly development is a critical period in fish aquaculture and is influenced by biotic and abiotic factors (e.g., temperature, feed) that can vary significantly between hatcheries, making it difficult to identify core factors determining quality. Many of the existing larval transcriptome studies are small-scale and occur under specific rearing conditions that do not mirror the diversity of larviculture practices at an industrial level. In the present transcriptome study, gilthead seabream at the larval to juvenile transition (metamorphosis) from several hatcheries in Europe (Greece, Italy, and France) were analysed in a large-scale RNA-seq study. The aim was to uncover the most significant molecular modifications occurring during metamorphosis, irrespective of differences in biotic or abiotic factors, to address knowledge gaps associated with critical early developmental stages under industrial hatchery conditions. Commonly modified gene transcripts between larval stages were identified based on the clustering of gene expression profiles of 25 gilthead seabream libraries from different hatcheries in a PCA analysis. When larvae at flexion were compared to larvae at mid-metamorphosis, 2243 differentially expressed genes (DEGs) were identified, and when larvae at early to mid-metamorphosis were compared to mid to late-metamorphosis, 2299 DEGs were identified. Comparative analysis across the developmental stages of gilthead seabream revealed genes of importance for the metamorphic transition and adaptation to rearing conditions, including genes related to the nervous system at flexion (24 days post hatch), enteroendocrine cell differentiation, and lipid homeostasis at early to mid-metamorphosis (46 dph), and enrichment of genes indicative of immune competence at mid to late-metamorphosis (51-54 dph). The differential expression of some endocrine-associated genes, dio1, dio2, cldn1, ing4, Pou3f4, and fgf22, highlights their importance in metamorphosis. Meta-analysis of the transcriptomes from two species, the gilthead seabream and Senegalese sole, that have differing symmetry and ecology uncovered common molecular expression patterns that underlie larvae maturation during metamorphosis, and we propose that these represent core gene markers of metamorphosis in these two fish species.
- Melatonin rhythms beyond the pineal organ: gene expression of receptors and biosynthesis enzymes in wild and F1 Senegalese solePublication . Félix de Azeredo Pinto e Melo, Francisca; Oliveira, Catarina; Martín, Ignacio; Manchado, Manuel; Sánchez-Vázquez, Francisco J.; Vera, Luisa M.; Cabrita, ElsaFish gonadal melatonin production is still unexplored and could contribute to a better understanding of its role in reproduction control, especially for species with reproductive impairments. This study aimed to comprehend if Senegalese sole testes are an extra-pineal production site of melatonin and if it has seasonal and daily variations. Wild and F1 broodstocks were sampled in the breeding season (BS) and out of the reproductive season (OS), at mid-light (ML) and mid-dark (MD) daytimes. Blood plasma melatonin concentration was determined by radioimmunoassay (RIA). The expression of genes involved in melatonin biosynthesis (tph1a, tph2, hiomt1, aanat1a, aanat1b, and aanat2) and melatonin receptors (mel1, mel1c, and mel2) was evaluated in the brain, eye, and testis by quantitative real-time PCR (qPCR). Plasma melatonin concentration in wild sole displayed day/night differences in both seasons (average ML: 36 +/- 22 pg/mL, MD: 108 +/- 63 pg/mL), whereas differences in the F1 broodstock were only found OS (ML: 100 +/- 54 pg/mL, MD: 187 +/- 88 pg/mL). Gene expression of mel1 and mel2 receptors, and tph1a, aanat1a, aanat2, and hiomt1 enzymes was detected and quantified in the fish testes. Moreover, daily and seasonal fluctuations in the expression of those genes were found in all tissues and broodstock groups. However, the F1 group showed distinct gene expression patterns compared to the wild type, suggesting a disruption in the circadian system. This study revealed that Senegalese sole testes are a melatonin production site and, at the same time, suggested a dysregulation in the hypothalamus-pituitary-gonad (HPG) axis of F1 males.
- Metamorphosis-associated immune system maturation in Senegalese solePublication . Silva, Sandra; Correia, Teresa; Manchado, Manuel; Power, Deborah MaryThe thyroid hormones (THs) are proposed as putative regulators of immune system maturation in developing teleost fish. To gain insight into this process the Senegalese sole (Solea senegalensis) that has a well-characterized TH-driven metamorphosis was used. Differential gene expression analysis was performed across developmental stages (n = 3 per stage): pre-metamorphosis, onset of metamorphosis, metamorphosis, early climax, climax, and post-metamorphic juveniles. Metamorphosis is a massive gene-oriented developmental process, involving the differential expression of 8145 genes. Clustering analysis was used to identify immune-related genes with similar expression patterns to hypothalamus-pituitary-thyroid (HPT) axis-related genes. TH-regulated candidate immune genes were identified (133) and analysis of their promoter region revealed 84 contained putative TH receptor (TR) binding sites (TREs). Two consensus TRE sequences were identified in the candidate genes, 5 '-ntgnGntCacan (exclusive to TR alpha), and 5 '-nnntGgtCannn (common to both TRs). TR alpha-exclusive TREs were less common than those that bound interchangeably TR alpha and TR beta. In the promoter region, TR alpha-exclusive TREs were always accompanied by the pan-TRE consensus sequence, never occurring independently.
- Microalgal extracts induce larval programming and modify growth and the immune response to bioactive treatments and LCDV in Senegalese sole post-larvaePublication . Carballo, Carlos; Mateus, Ana; Maya, Claudia; Mantecon, Lalia; Power, Deborah; Manchado, ManuelImmunostimulants are key molecules in aquaculture since they heighten defensive responses and protection against pathogens. The present study investigated the treatment of Senegalese sole larvae with a whole-cell crude extract of the microalgae Nannochloropsis gaditana (Nanno) and programming of growth and the immune system. Larvae at hatch were treated with the Nanno extracts for 2 h and thereafter were cultivated for 32 days posthatch (dph) in parallel with an untreated control group (CN). Dry weight and length at 21 days post-hatch (dph) were higher in post-larvae of the Nanno than CN group. These differences in weight were later confirmed at 32 dph. To evaluate changes in the immune response associated with Nanno-programming treatments, the Nanno and CN post-larvae were supplied with two bioactive compounds yeast beta-glucan (Y) and a microalga extract from the diatom Phaeodactylum tricornutum (MAe). The bioactive treatments were administrated to the treatment groups through the live prey (artemia metanauplii, 200 artemia mL(-1)) enriched for 30 min with MAe or Y (at 2 mg mL(-1) SW) or untreated prey in the case of the negative control (SW). The effect of the treatments was assessed by monitoring gene expression, enzyme activity and mortality over 48 h. The postlarvae sole supplied with the bioactive compounds Y and MAe had increased mortality at 48 h compared to the SW group. Moreover, mortality was higher in Nanno-programmed than CN post-larvae. Lysozyme and total antiprotease enzymatic activities at 6 and 24 h after the start of the trial were significantly higher in the Nanno and MAe supplied post-larvae compared to their corresponding control (CN and SW, respectively). Immune gene transcripts revealed that il1b, cxc10 and mx mRNAs were significantly different between Nanno and CN postlarvae at 6 and 24 h. Moreover, the expression of il1b, tnfa, cxc10, irf3, irf7 and mx was modified by bioactive treatments but with temporal differences. At 48 h after bioactive treatments, Y and SW post-larvae were challenged with the lymphocystis disease virus (LCDV). No difference existed in viral copy number between programming or bioactive treatment groups at 3, 6 and 24 h after LCDV challenge although the total number of copies reduced with time. Gene expression profiles in the LCDV-challenged group indicated that post-larvae triggered a wide defensive response compared to SWC 24 h after challenge, which was modulated by programming and bioactive compound treatments. Cluster analysis of expressed genes separated the SW and Y groups indicating long-lasting effects of yeast beta-glucan treatment in larvae. A noteworthy interaction between Nanno-programming and Y-treatment on the regulation of antiviral genes was observed. Overall, the data demonstrate the capacity of microalgal crude extracts to modify sole larval plasticity with long-term effects on larval growth and the immune responses.
- Olfactory sensitivity of the marine flatfish Solea senegalensis to conspecific body fluidsPublication . Fatsini, Elvira; Carazo, Ignacio; Chauvigne, Francois; Manchado, Manuel; Cerda, Joan; Hubbard, Peter; Duncan, Neil J.Chemical communication is better understood in freshwater fish than marine fish. The Senegalese sole (Solea senegalensis) is a marine flatfish wherein one of the problems in aquaculture is the poor reproductive performance of hatchery-bred males. Is chemical communication involved in the reproduction of this species? Urine, intestinal fluid and mucus samples were taken from adult fish (wild-caught and hatchery-bred) over the spawning season (March-May), and assessed for olfactory potency using the electro-olfactogram (EOG). The effect of stimulation of the olfactory system with adult female urine on circulating luteinizing hormone (LH) levels was also tested in males. Intestinal fluid and urine were potent olfactory stimuli for both juvenile and adult conspecifics, evoking large-amplitude, concentration-dependent EOG responses, with thresholds of detection at approximately 1:10(6). However, the amplitude of the response to urine depended on the sex and state of maturity of both the donor and the receiver. Most olfactory activity could be extracted by C18 solid-phase cartridges. Urine from mature females evoked a slight, but significant, increase in circulating LH levels in mature males 30 min after exposure. Furthermore, the olfactory potency of urine differed between wild-caught and hatchery-bred fish; however, contrary to expectations, urine from wild-caught females was less potent than that from hatchery-bred females. Taken together, these results strongly suggest that faeces-and urine-released odorants are involved in reproduction in the Senegalese sole, and establish a basis for further investigation into pheromonal communication in marine teleosts.
- Phylogeny, expression patterns and regulation of DNA Methyltransferases in early development of the flatfish, Solea senegalensisPublication . Firmino, Joana; Carballo, Carlos; Armesto, Paula; Campinho, Marco António; Power, Deborah M.; Manchado, ManuelBackground: The identification of DNA methyltransferases (Dnmt) expression patterns during development and their regulation is important to understand the epigenetic mechanisms that modulate larval plasticity in marine fish. In this study, dnmt1 and dnmt3 paralogs were identified in the flatfish Solea senegalensis and expression patterns in early developmental stages and juveniles were determined. Additionally, the regulation of Dnmt transcription by a specific inhibitor (5-aza-2 '-deoxycytidine) and temperature was evaluated. Results: Five paralog genes of dnmt3, namely dnmt3aa, dnmt3ab, dnmt3ba, dnmt3bb. 1 and dnmt3bb. 2 and one gene for dnmt1 were identified. Phylogenetic analysis revealed that the dnmt gene family was highly conserved in teleosts and three fish-specific genes, dnmt3aa, dnmt3ba and dnmt3bb. 2 have evolved. The spatio-temporal expression patterns of four dnmts (dnmt1, dnmt3aa, dnmt3ab and dnmt3bb. 1) were different in early larval stages although all of them reduced expression with the age and were detected in neural organs and dnmt3aa appeared specific to somites. In juveniles, the four dnmt genes were expressed in brain and hematopoietic tissues such as kidney, spleen and gills. Treatment of sole embryos with 5-aza-2 '-deoxycytidine down-regulated dntm1 and up-regulated dntm3aa. Moreover, in lecithotrophic larval stages, dnmt3aa and dnmt3ab were temperature sensitive and their expression was higher in larvae incubated at 16 degrees C relative to 20 degrees C. Conclusion: Five dnmt3 and one dnmt1 paralog were identified in sole and their distinct developmental and tissue-specific expression patterns indicate that they may have different roles during development. The inhibitor 5-aza-2 '-deoxycytidine modified the transcript abundance of dntm1 and dntm3aa in embryos, which suggests that a regulatory feedback mechanism exists for these genes. The impact of thermal regime on expression levels of dnmt3aa and dnmt3ab in lecithotrophic larval stages suggests that these paralogs might be involved in thermal programing.