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In keeping with the premises of Blue Circular Economy in the European Union, the present study explored the possibility of using the invasive brown alga Rugulopteryx okamurae in aquafeeds for European sea bass (Dicen-trarchus labrax). Assuming the raw algae biomass could negatively impact animal performance, four experi-mental formulations were prepared, by including macroalgae material at 5%, using crude (CR), enzymatically hydrolysed and fermented (EF), enzymatically hydrolysed (E), or fermented (F) R. okamurae biomass, which we tested against a control feed (CT). To evaluate the effects of the experimental diets, besides animal growth performance and biometric parameters, we devised a toolbox focused on the intestine and intestinal function: i) ex-vivo epithelial resistance and permeability in Ussing chambers; ii) microbiota composition through NGS; iii) expression profiles of selected markers for epithelial integrity, transport, metabolism, and immune response, by qPCR. Our results show differentiated allometric growth among diets, coupled with intestinal epithelium al-terations in permeability, integrity, and amino acid transport. Additionally, evidence of microbiota dysbiosis and contrasting immune responses between experimental diets, i.e. pro-inflammatory vs. anti-inflammatory, are also described. In conclusion, we believe that R. okamurae could be a suitable resource for aquafeeds for the European sea bass, although its use requires a pre-treatment before inclusion. Otherwise, while the fish still have a positive growth performance, the gastrointestinal tract pays a toll on the integrity, transport, and inflammatory processes. LA/P/0101/2020 info:eu-repo/semantics/publishedVersion

Stimulation and timing of smoltification are essential for successful Atlantic salmon (Salmo salar) aquaculture. This study investigated intestinal responses during dietary and photoperiod manipulation in freshwater (FW) and after a subsequent seven days residence in seawater (SW). "Small" and "large" Atlantic salmon parr (-40 g and -130 g respectively) were treated in FW for 12 weeks and thereafter transferred to SW for seven days. During the FW phase, fish underwent two different light conditions, 24 L:0D - 24 L ("LL-LL" groups) and 7 L:17D - 24 L ("SP-LL" groups) or fed with either regular feed ("LL-LL C" and "SP-LL C" groups) or feed enriched with a salt mix plus free tryptophan ("LL-LL + diet" and "SP-LL + diet" groups). We analyzed Na+/K+-ATPase (NKA) activity, tissue bioelectrical properties in Ussing chambers, and intestinal fluid composition. The NKA activity showed minor variations in relation to fish size, treatments, or intestinal region (anterior or posterior). Photoperiod modulated epithelial bioelectrical properties (Isc and Rt) of the anterior and posterior intestine, particularly transepithelial resistance (Rt). Pharmacological experiments, targeting apical Na+/K+/2Cl- (NKCC2) and Na+/ Cl (NCC) co-transporters revealed intestinal region-and water salinity-dependent effects. In addition, stimu-lation of the intracellular cAMP with forskolin and IBMX showed intestinal region-, water salinity, and treatment-dependence responses with clear functional specialization of the anterior and posterior intestine. The intestinal fluid composition reflected the ability to process ingested SW and showed little variation in large fish. In sum-mary, our data suggest a better pre-adaptation of the intestine during light-stimulated smoltification (SP-LL groups), and the combination of light and diet might give, in an industrial aquaculture setting, an advantage to smaller, but not larger smolts. Intestinal fluid composition in small fish can be used as an index of intestinal function and may act as a long-term performance proxy in SW Atlantic salmon. FHF - Norwegian Seafood Research Fund, Project no. 901432; LA/P/0101/2020 info:eu-repo/semantics/publishedVersion

Mycotoxins are an increasing threat to all the related commodities from agriculture. Its occurrence is expected to increase due to climate change. Here, we examined the impacts of dietary toxicity of aflatoxin B1 (AFB1) in gilthead sea bream (Sparus aurata) at levels of 1 or 2 mg AFB1 kg−1 fish feed. Inclusion of AFB1 in the diet resulted in 80% inhibition of the total weight gain during the 85-day trial. Carbohydrate and lipid energetic metabolites, both in plasma and liver, were depleted. Moreover, the histopathological analysis revealed several tissue anomalies in the liver, kidney, and spleen. Furthermore, the relative expression of gene transcripts for growth regulation was affected by AFB1. Adenohypophyseal gh and hepatic igf1 were inversely correlated due to AFB1 effects. Relative expression levels of gene transcripts as stress indicators were increased at AFB1 highest doses, such as hypothalamic trh, crh, and crhbp, as well as star in head kidney. Interestingly circulating levels of cortisol were unaffected. Overall, our results showed that aquafeeds with AFB1 impaired growth, alter metabolism, tissue integrity, and transcriptomic responses. However, we did find AFB1 residue neither in the liver nor muscle., This work was funded by the Spanish Ministry of Economy and Business-MINECO (AGL2016-76069-C2-1-R) awarded to JM.M. The authors (A.B., and J.M.M.) belong to the Fish Welfare and Stress Network (AGL2016-81808-REDT), supported by the Agencia Estatal de Investigación (MINECO, Spanish Government). A. Barany is currently supported by the University of Cadiz Ph.D. scholarship (PIF UCA/REC02VIT/2014). CCMar is supported by Portuguese national funds from the Foundation for Science and Technology (FCT) through project UIDB/04326/2020.

Post-smolt mortality and stunted growth is a problem in Norwegian salmon farming that can be partly traced back to sub-optimal smolt quality in connection with ongoing changes in smolt production practices. In the present study, we compared smolt development and post-smolt performance in seawater (SW) of Atlantic salmon subjected to 1) traditional light treatment (short photoperiod (SP) long photoperiod (LL)), 2) a dietary treatment alone (LL-LL + diet), 3) a combination of light and diet (SP-LL + diet) treatment and 4) no stimulation (LL-LL control) during the freshwater (FW) phase. The transition diet consisted of feed supplemented with a salt mixture and the amino acid tryptophan. Effects of light and dietary treatment were tested on two different size classes (40 and 130 g) of parr. Response patterns to treatments were independent of initial size and findings discussed below apply to both. Fish in the continuous light groups (LL-LL and LL-LL + diet) had the highest mass gain during the FW phase while light treated fish had a reduction in condition factor indicative of a true smolting. Hypoosmoregulatory ability in May was increased by diet, but not by light. However, a strong and treatment independent decrease in gill NKA alpha 1a gene expression indicated a complete shift from a FWto a SW type gill Na+, K+-ATPase. Moreover, plasma Cl- concentration and osmolality after 7 days in SW in May were well within the range expected for fully SW adapted fish in all treatment groups. Despite good hypoosmoregulatory abilities in all fish, there were strong treatment dependent effects on their growth during the two month SW residence. SW growth was negatively correlated with growth and change in K during the last part of the FW phase. A markedly higher specific growth rate and appetite in the SP-LL group than in the LL-LL group was likely a result of the light stimulated endocrine changes that occurred during smolting. Dietary treatment seemed to compensate for the lack of light treatment since feed intake and growth in LL-LL + diet fish was comparable to that in the SP-LL treated smolts. Feed intake and growth in the SP-LL + diet groups was highest, giving support for a notion that light and diet act through separate and additive mechanisms. FHF -Norwegian Seafood Research Fund [901432]; Portuguese Foundation for Science and Technology (FCT)Portuguese Foundation for Science and Technology [UID/Multi/04326/2019] info:eu-repo/semantics/publishedVersion

In 2019, Europe will adopt a no-discards policy in fisheries. This entails the landing of captured species unless strong evidence is provided supporting their survival and recovery after fishing. Thus, research on this topic is gaining momentum. Bottom-trawling, as a non-selective fishing method, is characterized by a high proportion of discards including vulnerable key species, such as demersal sharks. Their survival may also depend on capture depth. By paralleling onboard and laboratory experiments with the small-spotted catshark, Scyliorhinus canicula, we offer a robust experimental design to assess the survival of discarded sharks. Catsharks were captured by bottom-trawling at two depths (shallow similar to 89 m and deep similar to 479 m). Blood samples were collected following trawl capture and analyzed for stress biomarkers (lactate, osmolality, phosphate, urea). During recovery in onboard tanks, behavior was video-recorded and fish were re-sampled after 24 h. A second experiment was conducted in laboratory facilities to simulate air-exposure after trawling and to analyze the physiological recovery. Our results showed that 95.7% of the animals survived 24 h after trawling. We confirmed that trawling elicited acute stress responses in catshark but that they managed to recover. This was demonstrated by lactate concentrations that were 2.6 mM upon capture, but recovered to assumed baselines after 24 h (0.2 mM). Non-invasive video monitoring revealed behavioral differences with depth, whereby those captured at 89 m depth required longer to recover than those captured at 479 m depth. Implementation of standardized survival studies by fishery managers can benefit from holistic physiological approaches, such as the one proposed here. European UnionEuropean Union (EU) PADI Foundation [28467] Ministerio de Economia y CompetitividadSpanish Government [AGL2013-48835-C2-R, AGL2016-76069-C2-1-R] Project SUREDEPAR (Programa Pleamar, Ministerio de Agricultura y Pesca, Alimentacion y Medio Ambiente) Ministry of Science and Higher EducationMinistry of Science and Higher Education, Poland European Social Funds through the Portuguese National Science Foundation (FCT)Portuguese Foundation for Science and Technology [PTDC/MAR-BIO/3034/2014] FCTPortuguese Foundation for Science and Technology [UID/Multi/04326/2013]

Pituitary adenylate cyclase-activating polypeptide (PACAP) administered to tilapia melanophores ex-vivo causes significant pigment aggregation and this is a newly identified function for this peptide in fish. The G-protein coupled receptors (GPCRs), adcyap1r1a (encoding Pac1a) and vipr2a (encoding Vpac2a), are the only receptors in melanophores with appreciable levels of expression and are significantly (p

The dynamic changes in mRNA expression levels for vasotocin (AVT) and isotocin (IT) receptor gene levels were assessed in a time-course response study in immature male specimens of the gilthead sea bream (Sparus aurata) submitted to hyper- (55‰ salinity) and hypo-osmotic (5‰ salinity) challenges. Two different cDNAs for the AVT receptor and one for the IT receptor (V1a2-type and V2-type AVTR, and ITR, respectively) were cloned by screening an S. aurata brain cDNA library. Genes for these receptors were expressed differentially and is nearly ubiquitously in 26 of the examined tissues. In the gills, both environmental salinity challenges up-regulated AVTR V1a2-type gene expression concomitantly with mRNA expression protein activity of Na+, K+-ATPase gene expression and protein, whereas the AVTR V2-type and cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels were associated with mRNAs environmental salinity, indicating a possible connection between AVTRs and these transporters. In kidney, AVTR V1a2-type gene expression peaked rapidly and lasted only a short time (12-24h) in response to both osmotic challenges. In contrast, AVTR V2-type mRNA levels were enhanced in specimens exposed to hyperosmotic conditions, whereas they decreased under hypoosmotic environments, suggesting an antidiuretic role related to the vasoconstriction function. In the hypothalamus, only the expression of the AVTR V2-type gene was enhanced at 7 and 14days under both experimental conditions. In the liver, both AVTRs had increased mRNA levels, with the upregulation of their AVTR V2-type gene increasing faster than the V1a2-type. The ITR gene was not sensitive to variations of external salinity in any of the analyzed tissues. Our results demonstrate the involvement of the vasotocinergic, but not the isotocinergic, pathway as well as the hypothalamic function, in the adjustments of both osmoregulatory and metabolic processes after osmotic challenges. © 2013 Elsevier Inc., This study was funded by project AGL2010-14876 from the Ministry of Science and Innovation to J.M. Mancera (Spain), by AquagenomicsCSD2007-0002, and FEDER/ERDF. J.A. Martos-Sitcha waswas supported by a PhD fellowship (FPU, Reference AP2008-01194) from the Ministry of Education (Spain).

DAX1 is an orphan nuclear receptorwith actions in mammalian sex determination, regulation of steroidogenesis, embryonic development and neural differentiation. Conserved patterns of DAX1 gene expression frommammals to fish have been taken to suggest conserved function. In the present study, the European sea bass, Dicentrarchus labrax, DAX1 promoter was isolated and its conserved features compared to other fish and mammalian DAX1 promoters in order to derive common regulators and functional gene networks. Fish andmammalian DAX1 promoters share common sets of transcription factor frameworkswhichwere also present in the promoter region of another 127 genes. Pathway analysis clustered these into candidate gene networks associated with the fish and mammalian DAX1. The networks identified are concordantwith described functions for DAX1 in embryogenesis, regulation of transcription, endocrine development and steroid production. Novel candidate gene network partners were also identified, which implicate DAX1 in ion homeostasis and transport, lipid transport and skeletal development. Experimental evidence is provided supporting roles for DAX1 in steroid signalling and osmoregulation in fish. These results highlight the usefulness of the in silico comparative approach to analyse gene regulation for hypothesis generation. Conserved promoter architecture can be used also to predict potentially newgene functions. The approach reported can be applied to genes from model and non-model species. The authors acknowledge Heiner Kuhl and Richard Reinhardt for sequencing the sea bass DAX1 and DAX2 promoters and to Pedro Guerreiro for his help in the salinity transfer experiment. The research leading to these results was funded by European Community's Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 222719 - LIFECYCLE. RSTM and LAMD were in receipt, respectively, of fellowships SFRH/BPD/66742/2009 and PTDC/CVT/104750/2008 from the Portuguese Foundation for Science and Technology.

The intestine of marine fish plays a crucial role in ion homeostasis by selective processing of ingested fluid. Although arginine vasotocin (AVT) is suggested to play a role in ion regulation in fish, its action in the intestine has not been demonstrated. Thus, the present study investigated in vitro the putative role of AVT in intestinal ion transport in the sea bream (Sparus aurata). A cDNA encoding part of an AVT receptor was isolated and phylogenetic analysis revealed it clustered with the V1a2-type receptor clade. V1a2 transcripts were expressed throughout the gastrointestinal tract, from esophagus to rectum, and were most abundant in the rectum regardless of long-term exposure to external salinities of 12, 35 or 55p.p.t. Basolateral addition of AVT (10-6M) to the anterior intestine and rectum of sea bream adapted to 12, 35 or 55p.p.t. mounted in Ussing chambers produced rapid salinity and region dependent responses in short circuit current (Isc), always in the absorptive direction. In addition, AVT stimulation of absorptive Isc conformed to a dose-response curve, with significant effects achieved at 10-8M, which corresponds to physiological values of plasma AVT for this species. The effect of AVT on intestinal Isc was insensitive to the CFTR selective inhibitor NPPB (200μM) applied apically, but was completely abolished in the presence of apical bumetanide (200μM). We propose a role for AVT in the regulation of ion absorption in the intestine of the sea bream mediated by an absorptive bumetanide-sensitive mechanism, likely NKCC2. © 2013 Elsevier Inc., JAM-S is funded by Ministry of Education (Spain) through the program “Formación de Profesorado Universitario” (Ref: AP2008-01194). This work was partially supported by Ministry of Science and Education, Spain by Project AGL2010-14876 to JMM and by the Ministry of Science and Higher Education and European Social Funds through the Portuguese National Science Foundation by Project PTDC/MAR/104008/2008 to JF.

In previous in vitro studies, we have shown that the N-terminal region of parathyroid hormone-related protein (PTHrP) can stimulate cortisol production in sea bream, Sparus auratus, interrenal tissue, possibly through a paracrine action. In the current study, the systemic interaction between cortisol and PTHrP was studied in vivo. Sustained elevated blood cortisol levels, induced either by cortisol injection or confinement stress, suppressed circulating PTHrP 6 and 24-fold, respectively, by comparison to control fish. Dexamethasone treatment reduced cortisol levels, prevented the decrease of plasma PTHrP observed in confined fish and raised plasma PTHrP levels in non-confined fish. In contrast, a single injection of (1-34) PTHrP caused a short-term (within 30 min and up to 2.5 h) decrease in plasma cortisol. The antagonistic effects between PTHrP and cortisol were substantiated by an overall (data pooled from all experiments) highly significant negative correlation (r0=-0.745, p

The effect of estradiol-17b (E2) implants on the in vitro secretion of prolactin (PRL) and its modulation by vasoactive intestinal peptide (VIP) in a marine teleost, sea bream (Sparus aurata L.), was determined. Experiments were conducted during winter and spring. During winter, fish (n¼130, body weight 50–70 g) were randomly divided into 2 groups; control and E2 treated (10 mg/kg, wet weight). Fish were sacrificed after 7 days treatment and in vitro pituitary cultures in Ringer bicarbonate supplemented with increasing doses (0–200 nM) of VIP were carried out for 18 h. Culture medium was analysed by PAGE and secreted PRL quantified by densitometry. Fish treated with E2 secreted significantly more PRL ðP < 0:05Þ in vitro than control fish. In E2 primed fish VIP caused a dose-dependent inhibition of PRL secretion in vitro. VIP had no detectable effect on the secretion of PRL from control pituitaries. Treatment with E2 had a different effect during spring; PRL secretion was significantly decreased ðP < 0:01Þ compared with the control fish. Anatomical evidence of abundant VIPimmunoreactive nerve fibres in neurohypophysial (NH) tissue penetrating the rostral pars distalis provide further evidence supporting an action for VIPin the regulation of PRL cells. In conclusion, the responsiveness of PRL in the pituitary gland varied with season. Moreover, in the sea bream VIP appears to modulate PRL secretion from E2 primed pituitary glands. The present work was funded by the Fundac ~ao para a Ci^encia e a Tecnologia, Portugal (PraxisXXI 2/2.1/ BIA/211/94). J.F. and L.B. were funded by FCT Grants BPD/22033 and 4/5.3/PRODEP/2000, respectively. The authors thank the anonymous reviewers for their helpful critique.