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- Modulation of pituitary response by dietary lipids and throughout a temperature fluctuation challenge in Gilthead Sea BreamPublication . Sánchez-Nuño, Sergio; Silva, Sandra; Guerreiro, Pedro M; Ordóñez-Grande, Borja; Sanahuja, Ignasi; Fernández-Alacid, Laura; Ibarz, AntoniLow temperatures provoke drastic reductions in gilthead sea bream (Sparus aurata) activity and nourishment, leading to growth arrest and a halt in production. However, scarce data exist concerning the implications of central core control during the cold season. The aim of this work was to study the effects of low temperature and recovery from such exposure on the pituitary activity of sea bream juveniles fed 18% or 14% dietary lipid. A controlled indoor trial was performed to simulate natural temperature fluctuation (22 ◦C to 14 ◦C to 22 ◦C). Meanwhile, we determined the regulatory role of the pituitary by analyzing the gene expression of some pituitary hormones and hormone receptors via qPCR, as well as plasma levels of thyroidal hormones. In response to higher dietary lipids, hormone pituitary expressions were up-regulated. Induced low temperatures and lower ingesta modulated pituitary function up-regulating GH and TSH and thyroid and glucocorticoid receptors. All these findings demonstrate the capacity of the pituitary to recognize both external conditions and to modulate its response accordingly. However, growth, peripheral tissues and metabolism were not linked or connected to pituitary function at low temperatures, which opens an interesting field of study to interpret the hypothalamus–pituitary–target axis during temperature fluctuations in fish.
- Holothurians have a reduced GPCR and odorant receptor-like repertoire compared to other echinodermsPublication . Marquet, Nathalie; Cardoso, João CR; Louro, Bruno; Fernandes, Stefan; Silva, Sandra; Canario, AdelinoSea cucumbers lack vision and rely on chemical sensing to reproduce and survive. However, how they recognize and respond to environmental cues remains unknown. Possible candidates are the odorant receptors (ORs), a diverse family of G protein-coupled receptors (GPCRs) involved in olfaction. The present study aimed at characterizing the chemosensory GPCRs in sea cucumbers. At least 246 distinct GPCRs, of which ca. 20% putative ORs, were found in a transcriptome assembly of putative chemosensory (tentacles, oral cavity, calcareous ring, and papillae/tegument) and reproductive (ovary and testis) tissues from Holothuria arguinensis (57 ORs) and in the Apostichopus japonicus genome (79 ORs). The sea cucumber ORs clustered with those of sea urchin and starfish into four main clades of gene expansions sharing a common ancestor and evolving under purifying selection. However, the sea cucumber ORs repertoire was the smallest among the echinoderms and the olfactory receptor signature motif LxxPxYxxxxxLxxxDxxxxxxxxP was better conserved in cluster OR-l1 which also had more members. ORs were expressed in tentacles, oral cavity, calcareous ring, and papillae/tegument, supporting their potential role in chemosensing. This study is the first comprehensive survey of chemosensory GPCRs in sea cucumbers, and provides the molecular basis to understand how they communicate.
- 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.
- Transcriptomic analysis of differentially expressed genes in kidney and intestine of dicentrarchus iabrax fed different nutritional amounts of inorganic phosphatePublication . Vargas Lagos, Carolina Fabiola; Silva, Sandra; Guerrero, Laura; Oca, Marco Montes de; Louro, Bruno; Carvalho Alves, Alexandra; Rotllant, Josep; Guerreiro, Pedro MiguelPhosphorus (P), in the form of inorganic phosphate (Pi), is one of the most important macronutrients for all organisms, including fish. It is indispensable for the formation of hard tissues such as bones, but also for cell signalling and cell membrane formation, and energy transduction, among many other functions and is kept under well-controlled conditions, since its deficiency or overload may lead to skeletal malformation or ectopic calcification, disturbances of intermediary metabolism, growth and function impairment, endocrine dysfunction, and eventually death. Fish feeds used in aquaculture are therefore P-rich but excess/unused/excreted P in the effluents can lead to eutrophication and a consequent deleterious change in the aquatic ecosystem. The objective of this study was to evaluate the expression profiles and transcripts modified by dietary P, to identify pathways and mechanisms involved in P transport and regulation in the kidney and intestine. Juvenile Dicentrarchus labrax were fed using a commercial feed (1.1% P) or tailored-made feeds containing 0.05%, 1.1%, or 3% Pi. Fish (duplicate tanks, n = 10) were fed for 70 days and weighed periodically to evaluate growth changes. Kidney and intestine were used for RNA extraction. Next-Generation Sequencing and RNAseq library preparation were performed in an Illumina system following the manufacturer’s recommendations. Annotation was performed using the available sea bass genome assembly. Bioinformatic analysis showed significant differences in expression patterns among the three conditions tested in both tissues. In the kidney, increased P led to a total of 135 differentially expressed genes (DEGs; 82 up and 53 down), while only 54 (11 up and 43 down) genes responded to P restriction. In the intestine, high P affected the expression of 50 genes (16 up and 34 down) whereas only 26 (6 up and 20 down) were modified by low P. However, DEGs between high and low P were 156 in kidney and 154 in intestine. Preliminary analysis suggests the most affected pathways were those involved in cellular metabolism and phosphorylation but also on the structure of cell membranes, either for maintaining membrane integrity or in genes related to transmembrane ion transport. We expect this research to reveal the molecular implications of dietary P imbalance looking at specific targets such as membrane transporters and regulatory factors, but also to the larger metabolic pathways affected in these two key organs for P uptake and excretion.
- The stress response in antarctic fish: HPI modulation, cortisol profiles, interrenal sensitivity, and gene expression of notothenia rossii acclimated to temperature challengesPublication . Guerreiro, Pedro Miguel; Silva, Sandra; Louro, Bruno; Alves, Alexandra C.; Couto, Elsa; Canario, AdelinoThe Antarctic Ocean is one of the most extreme marine environments. Antarctic fishes evolved in stable cold thermal conditions (−1.9 ◦C to 2 ◦C) for roughly 20 million years, displaying structural and functional features resulting from adaptation or inherited from resilient ancestral species. Climate change forecast models show temperatures may increase at a relevant pace. As fish face a warmer future, their physiological ability to adapt is uncertain. We aimed at evaluating the capabilities of the hypothalamus–pituitary–interrenal (HPI) axis in Antarctic fish and show plasma cortisol profiles, expression of key genes, the sensitivity of the ex vivo interrenal tissue, and the responses to known modulators of the HPI axis in temperature-acclimated fish before and after stress. Notothenia rossii were collected from the waters of Admiralty and Maxwell bays, in King George Island, and transferred to an open circuit with ocean-pumped seawater. Upon acclimation, three sets of experiments were performed: (1) eight groups at 2 ◦C were injected with drugs involved in blockage or stimulation of cortisol release/action (saline, cortisol, dexamethasone, metyrapone, spironolactone, mifepristone) and then kept at control or transferred to 6 ◦C and sampled after 36 h; (2) fish at 2 ◦C were exposed to a standard stress test (SST: chasing+netting+1min air exposure), returned to the respective tank and sampled after 1, 4 and 24 h, while one undisturbed group served as control; (3) six groups were acclimated to 2, 5 and 8 ◦C for 10 days when the control group of each temperature was sacrificed. The other group received SST and was sacrificed 90 min after. Plasma and tissue samples were collected for cortisol and stress-related genes, and the interrenal was used in vitro to determine sensitivity to ACTH in a perfusion system with a continuous flow of oxygenated ringers, and 20 min fractions were collected for 240 min. Cortisol was measured via radioimmunoassay, while glucose and lactate were determined using colorimetric kits; gene expression was evaluated by qPCR. Manipulation of the HPI axis revealed that these fish show similar dynamics to those reported in temperate fish but with lower amplitude. After SST, cortisol peaked at 1–4 h and reduced to basal between 24 and 48 h. Temperature influenced the cortisol response to SST. At higher temperatures, cortisol levels in the non-stressed group were as high as in fish subjected to SST. Interrenal cells showed little response to ACTH in warm conditions, suggesting low sensitivity and/or exhaustion. Liver cortisol receptor genes were downregulated, possibly indicating a peripheral desensitization process that parallels HPI. These results show the ability to respond to stress at cold and mild temperatures but important impairments and substantial allostasis in warm or continuously increasing temperatures.