Search

Self-feeding and dualchoice systems have been extensively used to study animal feeding behaviour and food preferences but are still little explored in fish species. Seabream (Sparus aurata) is an important farmed Mediterranean species, but little is known regarding its taste preferences, including its capacity to discriminate diets using sensory (olfactory and/or gustative) traits. With this in mind, the capability of gilthead seabream to discriminate feed organoleptic qualities was investigated by presenting the fish with a dual choice of feeds in a self-feeding system. Furthermore, two consecutive trials were performed with the same group of fish during summer or autumn (i.e., at different water temperatures), to assess whether the feeding behavior might be affected by the fishes' physiological state or environmental parameters.

Trabajo presentado en el XVIII Congreso nacional de acuicultura, celebrado en Cádiz (España) del 21 al 24 de noviembre de 2022.

Resumen del trabajo presentado en Aquaculture Europe 2022, celebrado en Rimini (Italia) del 27 al 30 de septiembre de 2022., [Introduction]: Taste receptors (TRs) are classically associated to the perception of gustatory sensations in oral tissues. However, many TRs are also found in different body tissues including the gastrointestinal tract (GIT), where they perform important roles (Finger and Kinnamon, 2011). In the GIT, the presence of TRs in specialized cells (enteroendocrine cells: EECs) regulates digestive, absorptive and metabolic functions through gut sensing mechanisms (Alpers, 2010). Functional actions of TRs are associated to their co-expression with gut peptides, including ghrelin (GHR), cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1) or peptide YY (PYY), to cite a few. Once a TR is activated, an intracellular signaling cascade is initiated, terminating with peptide secretion in the EEC basolateral side, which can signal neighboring (paracrine action) or remote (endocrine action) cells, or activate afferent neurons (neural action). The taste receptor type 1 (T1R) family is one of the TR types that is commonly expressed in EECs, acting as heterodimeric sensors of nutrients in the mammalian gut: T1R1/ T1R3 responding to umami compounds such as amino acids, and T1R2/T1R3 to sweet substances. We previously showed the expression of the Sparus aurata (sa) T1R gene repertoire in oropharyngeal, GIT and brain tissues. However, this does not necessarily imply a conserved role in gut sensing. Furthermore, expression in GIT was observed in early larval stages but no expression was found in oropharyngeal tissues up to 12 days post-hatching (dph), which was puzzling. The present study aimed to provide direct evidence for mRNAs co-expression of saT1R genes (mostly t1r3, as the common element of both receptors) with EEC-type peptides such as ghr, cck, pyy and proglucagon (pg), to establish a morphological link indicating possible roles of T1R in gut nutrient-sensing mechanisms and in the regulation of fish digestive processes. A second objective was to extend the period of investigation during larval ontogeny to establish the temporal pattern of expression in GIT and oral tissues for the entire saT1R gene repertoire in relation to first-feeding.

Trabajo presentado en el XX International symposium on fish nutrition and feeding towards precision fish nutrition and feeding, celebrado en Sorrento (Italia) del 05 al 09 de junio de 2022.

9 `pages, 5 figures, 3 tables, The melanocortin system is an ancient neuroendocrine system conserved from teleosts to mammals. The melanocortin system is a set of complex neuroendocrine signaling pathways involved in numerous physiological processes, and particularly associated with the hypothalamic-pituitary-interrenal (HPI) axis response. The melanocortin 1 receptor (MC1R) is the central melanocortin receptor involved in pigmentation in vertebrates, including fish. In order to assess the immune role of MC1R, this study used a homozygous Mc1r knockout zebrafish. Hence, skin cortisol levels, variations in the blood leucocyte population, as well as the expression levels of immune genes in various tissues of wild-type TU strain (Tübingen, Nüsslein-Volhard Lab) (WT) and homozygous mc1r knockout zebrafish (mc1rK.O.) stimulated with LPS was carried out. Results show that the mc1rK.O. mutant fish produce lower levels of cortisol in mucus and fewer macrophages in blood after exposure to LPS compared to control fish. Regarding the expression of immune genes, mutant fish show a significant increase in the expression of the anti-inflammatory interleukin il10. These results suggest that the mc1rK.O. mutant fish may follow an alternative mechanism among the immune responses, where macrophages seem to have an anti-inflammatory function, attenuating nitric oxide (NO) production and providing an advantage through the mitigation of excessive or strong inflammatory reactions. Nonetheless, a lower number of this cell type could imply a reduced phagocytic potential in the face of an infection. At the same time, lower cortisol levels in the mc1rK.O. mutant fish could be an advantage as for the lower susceptibility to stress and the physiological and metabolic consequences of high cortisol levels, Part of this work was funded by grant number AGL2017-89648P from the MCIN/AEI/10.13039/501100011033 and by ERDF A way of making Europe

Trabajo presentado en el XVIII Congreso Nacional de Acuicultura, celebrado en Cádiz (España) del 21 al 24 de noviembre de 2022., [EN]: Overexpression of agouti-signalling protein (ASIP) in transgenic zebrafish increases food intake levels and linear growth. A higher feed intake is unnecessary in transgenic fish to enable larger and heavier growth. A plausible explanation may rely on the enhanced feeding efficiency mediated by improved nutrient absorption in transgenic animals. Transcriptomic results suggested that amino acid, monocarboxylates, ionic and vitamin transmembrane transport was substantially modified. Enrichment analysis revealed an inhibition of intestinal lipid metabolism and down-regulation of KEGG pathways related to membrane integrity suggesting an augmented intestinal laxity that may result in enhanced paracellular transport. Electrophysiological experiments showed that asip1 overexpression decrease membrane tissue resistance (Rt), indicating a modification of the intestinal barrier function in ASIP1 transgenic animals. Paracellular permeability was higher in ASIP fish. Both the decrease in Rt and the increase in permeability point to an ASIP1-dependent decrease in the tissue barrier function. Electrogenic amino acid transport was enhanced in transgenic fish providing a strong indication that ASIP1 fish can extract more amino acids from their diet at similar feeding levels. Both transcriptomic and electrophysiological results support that asip1-overexpressing zebrafish display improved nutrient absorption and by extension a higher feed efficiency which explains enhanced growth in the absence of augmented food intake., [ES]: La sobreexpresión de la proteína de señalización agoutí (ASIP) en peces cebra transgénicos incrementa los niveles de ingesta y el crecimiento, además, los peces ASIP1 no necesitan comer más para crecer más. Una posible explicación de lo anteriormente expuesto, podría ser una mejora de la eficiencia alimentaria mediada por una absorción de nutrientes más eficaz en los peces ASIP1. Para comprobar esta hipótesis realizamos dos tipos de experimentos. Por un lado, realizamos una comparación de la transcriptómica intestinal de ambos genotipos y subsecuentemente analizamos la integridad y transporte intestinal mediante el uso de cámaras Ussing, Los resultados transcriptómicos sugieren que el transporte de membrana de aminoácidos, monocarboxilados, iones y vitaminas está sustancialmente modificado. El enriquecimiento de algunos genes muestra una inhibición del metabolismo lipídico intestinal y una disminución en la regulación de las vías KEGG relacionadas con la integridad de membrana, apuntando a un incremento de la laxitud intestinal, que quizá interfiera en el transporte paracelular. Experimentos electrofisiológicos mostraron que la sobreexpresión de asip1 redujo la resistencia de la membrana (Rt) y acrecentó la permeabilidad paracelular, revelando una modificación de la función de la barrera intestinal en los peces ASIP1. El transporte electrogénico de aminoácidos se vio incrementado en los peces transgénicos sugiriendo que los peces ASIP1 pueden extraer más aminoácidos de la dieta con similares niveles de ingesta. Los resultados transcriptómicos y electrofisiológicos apoyan la idea que la sobreexpresión de asip1 mejora la absorción de nutrientes y por ende la eficiencia alimentaria, lo cual explicaría el mayor crecimiento obtenido en ausencia de necesitar mayores cantidades de alimento en los peces transgénicos., Proyecto PID2019-103969RB-C33 de la Agencia Estatal de Investigación (AEI).

Poster.-- XVIII Congreso Nacional de Acuicultura “Acuicultura: mares y ríos de oportunidades”, Cádiz, del 21 al 24 de noviembre de 2022, [EN] The agouti-signaling protein (ASIP) acts as a competitive antagonist and inverse agonist of melanocortin receptors which regulate dorsal-ventral pigmentation patterns in fish. However, the role of ASIP in the regulation of additional physiological pathways in the skin is unknown. The skin plays a crucial role in the immune function, acting as a physical limitation and also as a chemical barrier due to its ability to secrete mucus. In this study, the putative role of ASIP in skin immune response has been explored using a transgenic zebrafish overexpressing the asip1 gene (ASIPzf). The structural changes in skin induced by asip1 overexpression revealed that the ventral skin of ASIPzf was thinner than that of wild type (WT) animals. Histochemical studies showed that transgenic animals appear to compensate for the lower number of cell layers by modifying the mucus composition and increasing lectin affinity and mucin content in order to improve protection against microorganism adhesion. ASIPzf also exhibit higher protein concentration under crowding conditions suggesting an increased mucus production under stressful conditions. Exposure to bacterial lipopolysaccharide (LPS) showed that ASIPzf exhibit a faster pro-inflammatory response and increased mucin expression. Electrophysiological measurements show that the ASIP1 genotype exhibits reduced epithelial resistance, an indicator of reduced tissue integrity and barrier function. Overall, not only are ASIP1 animals more prone to infiltration and subsequent infections due to reduced skin epithelial integrity, but also display an increased inflammatory response that can lead to increased skin sensitivity to external infections, [ES] La proteína de señalización agoutí (ASIP) actúa como antagonista endógeno de los receptores de melanocortinas y regula el patrón dorsoventral de pigmentación en peces (Ceinos et al., 2015), aunque sus funciones en la regulación de otros procesos fisiológicos de la piel se desconocen. La piel es fundamental en la respuesta inmune, actuando como barrera física y química, mediante la secreción de mucus. En este estudio, se ha analizado el papel de ASIP en la respuesta inmune de la piel, mediante un pez cebra transgénico que sobreexpresa asip1 (ASIPzf). Los cambios estructurales inducidos por dicha sobreexpresión, muestran que la piel ventral de los ASIPzf es más delgada que la de los peces salvajes (WT). Estudios histoquímicos revelan que los peces transgénicos parecen compensar la reducción en las capas epiteliales modificando la composición del mucus e incrementando la afinidad por lectinas y mucinas para mejorar la protección frente a la adhesión de microorganismos. Además, ASIPzf presentan mayor concentración de proteínas en el mucus tras condiciones de estrés por densidad, sugiriendo un incremento de la secreción de mucus durante estas condiciones. Tras la exposición a un lipopolisacárido bacteriano (LPS), ASIPzf presentan una respuesta pro-inflamatoria más rápida y mayor expresión de mucinas. Análisis electrofisiológicos manifiestan menor resistencia epitelial en ASIP1, un indicador de la reducción de la integridad del tejido y su función como barrera. En general, no solo los ASIP1 son más susceptibles a infecciones como consecuencia de la reducción epitelial, sino que incrementan la respuesta inflamatoria pudiendo aumentar la sensibilidad a infecciones externas, Proyecto PID2019-103969RB-C33 de la Agencia Estatal de Investigación (AEI)

13 pages, 14 figures, In two swordtail species of the genus Xiphophorus, onset of puberty in males and females, fecundity in females, and adult size in males are modulated by sequence polymorphism and gene copy-number variation at the P locus affecting the type 4 melanocortin hormone receptor (Mc4r). The involvement of Mc4r in regulating the onset of puberty outside the genus Xiphophorus remains unclear. In this study we used a transgenic line overexpressing asip1 (asip1-Tg), an endogenous antagonist of both type 1 melanocortin hormone receptor (Mc1r) and Mc4r, to investigate the relevance of the melanocortin system on the onset of puberty and adult reproductive performance in zebrafish (Danio rerio). Comparison of growth, puberty and reproductive performance between wild-type (WT) and asip1-Tg zebrafish revealed that a decreased activity of the melanocortin system did not change the timing of puberty but significantly delayed early growth of transgenic animals. Hatching time was postponed in asip1-Tg fish and they were significantly smaller than their WT siblings at 75 dpf, despite showing enhanced linear growth after having completed puberty. asip1-Tg females produced 1.38 times more eggs but spawned less frequently, and their eggs had showed a 0.89-fold smaller diameter but a 1.04-fold increase in larvae body length at hatching. Therefore, we demonstrate that asip-tg zebrafish do not reach puberty earlier than WT counterparts as it could be expected considering the enhanced length and weight growth during early adulthood. This is so because the effects of transgene on growth are noticeable after a threshold length, when puberty has already been reached. Data show that the inhibition of melanocortin system via asip overexpression is an excellent strategy to promote growth, in absence of obesity, by enhancing food efficiency but without accelerating puberty timing. Data here presented provides a deeper characterization of the phenotype induced by the decreased activity of the melanocortin system in fish thus providing an excellent strategy for future aquaculture especially because U.S. Food and Drug Association has recently approved transgenic fish trading, This research was funded by Spanish State Agency of Research (AEI), grant number AGL2016-74857-C3-3-R and PID2019-103969RB-C33 to JMCR and AGL2017-89648P to JR, Science and Technology Foundation (FCT, Portugal), grant number PTDC/CVT-CVT/3205/2020 to AR and National Agency for Research and Development (ANID), Scholarship Program, DOCTORADO BECAS CHILE fellowship 2013–72140242 to SN

Trabajo presentado en Aquaculture Europe, celebrado en Berlín (Alemania) del 7 al 10 de octubre de 2019., [Introduction]: Taste perception of sweet and umami stimuli in vertebrates is largely controlled by a family of G-protein-coupled receptors, the taste receptor type 1 (T1R) family, with three members that combine in heterodimers to form the taste receptors. The mammalian T1R1/T1R3 receptor responds to umami compounds such as amino acids, whereas T1R2/T1R3 is activated by sweet substances. In mammals, it has been well established that taste sensing in the oral cavity (taste buds) not only guides the consumption of foods, but the presence of taste receptors in specialized cells of the gastrointestinal tract (enteroendocrine cells) can also regulate digestive, absorptive and metabolic functions through gut sensing mechanisms (Alpers, 2010; Depoortere, 2014). In fish, however, very little information is available, particularly in species of aquaculture interest. We have previously cloned the full-length cDNA of five T1R genes in Sparus aurata (sa), including the specific heterodimer subunit of the umami taste (saT1R1), three novel sweet taste duplicate genes (saT1R2x, saT1R2y, saT1R2z) and the saT1R3 gene common to both umami and sweet taste heterodimers. The objective of this study was to further characterize their functional properties and patterns of expression in both larval and adult seabream tissues., [Materials and methods]: In order to functionally characterize this family of receptors we developed a stable Ca2+/reporter gene activity cell line assay (NFAT-Luc-HEK-293) to specifically examine the responses of the multiple saT1R(s) heterodimers to amino acid ligands. On the other hand, gene expression of four T1R genes (saT1R2x is not expressed, being likely a pseudogene) was examined through qPCR on different adult tissues to assess possible differences in the profile of tissue distribution. A qPCR analysis was also performed in whole larval tissues from 1 until 12 days post-hatching (dph), spanning life-stage transition from yolk-sac sustenance to exogenous feeding (initiated at 9 dph). To establish the tissue profile of gene expression in larvae, whole-mount in situ hybridization (WISH) was also performed to localize the expression of the four genes in preand post-exogenous feeding larvae (5 and 12 dph, respectively)., [Results and discussion]: Cotransfections of the reporter cell line with saT1R1/R3, saT1R2z/R3 and saT1R2y/R3 heterodimers showed a doseresponse activation of the putative (mammalian-homologous) umami and the two sweet receptors by several amino acid stimuli suggesting that, unlike mammals but similarly to what was previously reported in zebrafish and medaka (Oike et al., 2007), the three taste subunits may equally serve to transduce umami/amino acid taste sensations. Furthermore, the in vitro assays evidenced a fairly promiscuous activation profile of the three heterodimers, although with some differences in the sensitivity to some amino acids (not shown). Gene expression in adult tissues (figure 1) showed a predominant expression in oral tissues including lips (L), tongue (T) and oral cavity (OC) for all genes, and in particular for saT1R3, in agreement with its common involvement in the three receptor types. However, all the subunits were also found in gills (G), stomach (ST), foregut (FG), midgut (MG), hindgut (HG), forebrain (FB), midbrain (MB) and hindbrain (HB) tissues, even if with different profiles that might indicate a functional specialization. Quantitative saT1R(s) mRNA expression in whole seabream larvae (figure 2) showed that the four transcripts are stably expressed from 1 dph onwards and greatly increase shortly after exogenous feeding (initiated at 9 dph). WISH (figure 3) reveals the lack of expression of saT1R(s) in oral tissues of both pre- (5 dph) and post- (12 dph) exogenous feeding stages, with the genes being expressed exclusively in the gastrointestinal tract. These findings indicate an adaptive evolution of saT1R gene repertoire towards amino acid perception, and provide a molecular/cell basis for both oral and gut chemoreception in seabream. Surprisingly, however, results in young larvae suggest only gut sensing capabilities. Studies are underway to determine the ontogenetic onset of saT1R expression in oral taste sensing tissues, by examining later larval stages.

The melanocortin-4 receptor (MC4R) can be endogenously activated by binding of melanocyte-stimulating hormones (MSH), which mediates anorexigenic effects. In contrast, the agouti-related peptide (AgRP) acts as an endogenous inverse agonist and suppresses ligand-independent basal signaling activity (orexigenic effects). Binding of ligands to MC4R leads to the activation of different G-protein subtypes or arrestin and concomitant signaling pathways. This receptor is a key protein in the hypothalamic regulation of food intake and energy expenditure and naturally-occurring inactivating MC4R variants are the most frequent cause of monogenic obesity. In general, obesity is a growing problem on a global scale and is of social, medical, and economic relevance. A significant goal is to develop optimized pharmacological tools targeting MC4R without adverse effects. To date, this has not been achieved because of inter alia non-selective ligands across the five functionally different MCR subtypes (MC1-5R). This motivates further investigation of (i) the three-dimensional MC4R structure, (ii) binding mechanisms of various ligands, and (iii) the molecular transfer process of signal transduction, with the aim of understanding how structural features are linked with functional-physiological aspects. Unfortunately, experimentally elucidated structural information is not yet available for the MC receptors, a group of class A G-protein coupled receptors (GPCRs). We, therefore, generated MC4R homology models and complexes with interacting partners to describe approximate structural properties associated with signaling mechanisms. In addition, molecular insights from pathogenic mutations were incorporated to discriminate more precisely their individual malfunction of the signal transfer mechanism., This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategies—EXC 314/2 (UniCat to GK and PS) and EXC 2008/1 (UniSysCat)−390540038 (to PS). JC-R was funded by AGL2016-74857-C3-3-R. LS was supported by pre-doctoral fellowship FPI funded by Spanish Economy and Competitiveness Ministry. This work was further supported by the Deutsche Forschungsgemeinschaft SPP1629 BI893/5-2, KR1701/5-1 (to HB and HK), SFB740-B6 (to PS), SFB1078-B6 (to PS), and SFB1365-A03 (to PS).

Proopiomelanocortin (POMC) belongs to the opioid/orphanin gene family whose peptide precursors include either opioid (YGGF) or the orphanin/nociceptin core sequences (FGGF). In addition to POMC the family includes the proenkephalin (PENK), prodynorphin (PDYN), and nociceptin/proorphanin (PNOC) precursors. The opioid core sequence in POMC is incorporated by the β-endorphin that occupies the C-terminal region but this propeptide also exhibits at least two “alien” melanocortin core sequences (HFRW). An ACTH/MSH fragment merged into the opioid fragment not earlier than the two tetraploidizations of the vertebrate genome. Therefore, POMC exhibit a complex “evolutionary life” since the gene has coevolved together with two different receptor systems, i.e., opioid and melanocortin following a horse trading system. In this article, we summarize the different evolutionary hypotheses proposed for POMC evolution.

XIII Congress of the Iberian Association of Comparative Endocrinology - AIEC (online), 16-17 September 2021, In two swordtail species of the genus Xiphophorus, onset of puberty in males and females, fecundity in females, and adult size in males are modulated by sequence polymorphism and gene copy-number variation at the P locus affecting the type 4 melanocortin hormone receptor (Mc4r). The involvement of Mc4r in regulating the onset of puberty outside the genus Xiphophorus remains unclear. Mc4r binds the melanocyte-stimulating hormones (Mshs) and two inverse agonists, agouti-related protein (Agrp) and agouti-signalling protein (Asip). Both Agrp and Asip inhibit the constitutive activity of Mc4r and antagonistically compete with α-Msh. We used a transgenic line overexpressing asip1 (asip1-Tg), to investigate the relevance of the melanocortin system on the onset of puberty and adult reproductive performance in the zebrafish (Danio rerio). By comparing wild-type (WT) and asip1-Tg zebrafish we show that a decreased activity of the melanocortin system does not change the timing of puberty but significantly delays early growth of transgenic animals. Hatching time is postponed in asip1-Tg fish and they are significantly smaller than their WT siblings at 75 dpf, despite showing enhanced linear growth after having completed puberty. Even though asip1-Tg females spawn less frequently they produce 1.38 times more eggs that are smaller in diameter but render larvae with a 1.04-fold increase in body length at hatching. We demonstrate that the decreased activity of the melanocortin system induced by asip overexpression does not accelerate the puberty timing but significantly delays early growth of transgenic animals. Once asip-tg animals have outperformed a threshold length, when puberty has already been reached, the transgene rapidly promotes linear growth in absence of obesity, Funded by MINECO grants (AGL2016-74857-C3-3-R, PID2019-103969RB-C33 and AGL2017-89648P), FCT (PTDC/CVT-CVT/3205/2020) and National Agency for Research and Development (2013–72140242).

XIII Congress of the Iberian Association of Comparative Endocrinology - AIEC (online), 16-17 September 2021, The melanocortin complex system is composed of agonists, antagonists, several receptors, and accessory proteins. They are expressed in different tissues and participate in several physiological functions. The endogenous antagonist, agouti-signaling protein (ASIP), binds MC1R and MC4R decreasing their constitutive activities to regulate skin pigmentation and energy expenditure, respectively. Overexpression of ASIP1 in zebrafish (Danio rerio) disrupts the dorsoventral pigmentation pattern and enhances growth yet no obesity. Melanocortin system is also involved in the regulation of fish behavior. ASIP1 transgenic fish show higher levels of anxiety-related behaviour, are sight, display reduced exploratory behaviour, and are less aggressive than wild-type fish yet no less active. For that reason, we were interested in evaluating circadian rhythms (LCRs). During a month, we measure the locomotor activity of both wild type and ASIP1 transgenic fish using a custom system composed of photosensitive cells connected to Automata software. Data were analyzed with “El Temps” software that also allows creating chronograms and periodograms. Chronograms showed heightened locomotor activity in ASIP1 fish during the dark phase, and periodograms revealed the absence of circadian periodicity in locomotor activity in these fish. The brain and liver of 10 animals were dissected every 4 hours for a period of 24 hours on order to evaluate the daily expression patterns of clock related genes including: cry1a, per1a, clock1a, bmal1 and nr1d1. All three per1a, clock1a and nr1d1 showed enhanced expression levels during the dark phase of the photoperiod in ASIP1 fish. On the contrary, bmal1 expression ASIP1 fish raised coinciding with the lights turned on and during the light phase. These findings provide evidence for the role of the melanocortin system in the regulation of circadian rhythms, We are gratefully financed by funds from the Spanish Ministry of Economy and Competitiveness (AGL2017-89648P to J.R. and AGL2016-74857-C3-3-R, PID2019-103969RB-C33 to J.M.C.-R.). AG-G was funded by FPI program (BES-2017-082424) of the Spanish State Agency of Research (AEI)

XIII Congress of the Iberian Association of Comparative Endocrinology - AIEC (online), 16-17 September 2021, The melanocortin system is one of the most complex hormonal systems in vertebrates. The main melanocortin peptides are the melanocyte-stimulating hormones (MSHs) and adrenocorticotropic hormone (ACTH), all of them encoded by a common precursor named proopiomelanocortin (POMC), which is expressed mainly in the pituitary. Melanocortin signaling is mediated by a family of G protein-coupled receptors. In tetrapod species, five different subtypes (MC1R-MC5R) has been characterized yet number diverges in fish species. Melanocortin signaling is mediated also by endogenous antagonists which compete with melanocortin agonist by binding to MCRs. Agouti-signaling protein (ASIP) is mainly expressed in the skin where compete by binding to MC1R whereas agouti-related protein (AGRP) is expressed in the hypothalamus to regulate energy balance by binding central MC3R and MC4R. The skin plays a crucial role in the immune function, working as a physical impediment against infestation yet also as a chemical barrier through its ability to synthesizes and secretes mucus. In addition, the skin can be considered also as part of the adaptive immune response characterized by a much more specific response to pathogens. The melanocortin system has been described to regulate inflammatory process which constitutes a key point in the defense response against the damage factors and it is an important component of the innate immune system. Our recent studies using a zebrafish strain overexpressing ASIP1 have demonstrated the participation of the melanocortin system in the regulation of skin physiology. This transgenic model thus constitutes a key opportunity to evaluate the role of the melanocortins in the skin immune response. Our results show that transgenic fish exposed to lypopolysaccharide (LPS) are capable to increase significantly the mRNA levels of pro-inflammatory skin cytokines, suggesting better immune response than wild type fish against bacterial infections, Founded by Spanish Science and Innovation Ministry, project PID2019-103969RB-C33.

XIII Congress of the Iberian Association of Comparative Endocrinology - AIEC (online), 16-17 September 2021, The melanocortin system is a complex neuroendocrine signaling mechanism involved in numerous physiological processes in vertebrates, including pigmentation, steroidogenesis and metabolic control. In humans, five melanocortin receptors (MCR) have been cloned, identified and shown to have a wide distribution throughout the body and likely many diverse functions. It has also been shown that a high degree of identity and conservation in structural characteristics and pharmacology exists between Mcrs from fish and mammals. However, in fishes, the number, affinity, specificity, tissue distribution and physiological roles are far from defined and appear to be species-specific. A clear example is the Mcr2 subtype. In humans, it is well known that Mc2r is expressed in the adrenal gland and controls steroidogenesis and the same function has also been described in fish. In human, ACTH overproduction results into hyperpigmentation because of pharmacological effects on MC1R. Using CRISPR/Cas9, we demonstrated that zebrafish lacking Mc2r shows hyperpigmentation. Ongoing studies are exploring potential direct/indirect roles for Mc2r in fish skin pigmentation, This work was funded by the Spanish Economy and Competitiveness Ministry project AGL2017-89648P to JR

Aquaculture Europe 2021, Funchal, Madeira, 4-7 October, The melanocortin system is a complex neuroendocrine signaling mechanism involved in numerous physiological processes in vertebrates, including pigmentation, steroidogenesis and metabolic control. In humans, five melanocortin receptors (MCR) have been cloned, identified and shown to have a wide distribution throughout the body and likely many diverse functions. It has also been shown that a high degree of identity and conservation in structural characteristics and pharmacology exists between Mcrs from fish and mammals. However, in fishes, the number, affinity, specificity, tissue distribution and physiological roles are far from defined and appear to be species-specific. A clear example is the Mcr2 subtype. In humans, it is well known that Mc2r is expressed in the adrenal gland and controls steroidogenesis and in fact the same function has also been described in fish. However, the fact that Acth (a melanocortin agonist) may have a role in regulating fish pigmentation and that Acth acts on Mc2r, the involvement of Mc2r in fish pigmentation might be a possibility. Using CRISPR/Cas9 genome engineering tools we have generated “loss-of-function” mc2r mutant fish. We demonstrate that Mc2r , apart from controlling steroidogenesis, also has a direct role in regulating fish pigmentation

Trabajo presentado en el 18th International Symposium on Fish Nutrition and Feeding (ISFNF), celebrado en Las Palmas de Gran Canaria (España), del 3 al 07 de junio de 2018, Over the past decade, sustainable aquaculture production promoting increasing dietary replacement of fisheries by plant ingredients has steadily grown, often reducing the palatability of fish diets and animal performance. However, very little information exists on taste-related mechanisms possibly underlying these effects. Vertebrate taste perception is largely controlled by two families of G-protein-coupled receptors, taste receptor 1 (TAS1R) and taste receptor 2 (TAS2R), involved in recognition of sweet/umami and bitter tastes, respectively. Tastant selectivity through the TAS1R family is determined by the nature of combinatorial arrangement of TAS1R1, TAS1R2 and TAS1R3 heterodimeric subunits. The mammalian TAS1R1/TAS1R3 receptor responds to umami compounds such as amino acids, whereas TAS1R2/TAS1R3 is activated by sweet substances. Nevertheless, in some teleost fish, TAS1R2 duplicated genes revealed species-specific functional plasticity with a prominent response to amino acid ligands. In order to investigate TAS1R functional properties and their potential distinct evolutionary trajectories in fish, we cloned the full-length cDNA of five TAS1R genes in Sparus aurata (sa), including the specific heterodimer subunit of the umami taste (saTAS1R1), three novel sweet taste duplicate genes (saTAS1R2x, saTAS1R2y, saTAS1R2z) and the saTAS1R3 gene common to both umami and sweet taste heterodimers. Gene expression analyses indicate that, although to a different extent, saTAS1R1, saTAS1R2z, saTAS1R2y and saTAS1R3 genes are expressed in oral, gut and brain tissues. Interestingly, the absence of saTAS1R2x mRNA expression in all tissues investigated and the strikingly high levels of saTAS1R2y gene expression in oral and gut tissues indicate that saTAS1R2 paralogs may be under differential evolutionary pressure and may show functional specialization. Furthermore, we identified and cloned two G-protein alpha subunit genes (saG(i)α1 and saG(i)α2) putatively involved in the first steps of intracellular taste signaling in fish. Heterologous transfection studies on multiple saTAS1R heterodimers coupled to saG(i)α1 or saG(i)α2 have just begun to be performed and will contribute to advance important knowledge on fish tastephysiology. This study provides an essential groundwork to further elucidate the evolution and functional roles of the TAS1R gene repertoire in seabream, to ultimately develop products enhancing the attractability of fish diets and possibly also regulating digestive/absorptive functions and metabolism through gut sensing mechanisms

Trabajo presentado en el 11º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Vigo (España), del 13 al 15 de julio de 2017

Trabajo presentado en el 11º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Vigo (España), del 13 al 15 de julio de 2017, Regulation of pubertal timing remains unresolved in most species including humans. However the first sexual maturation in Xiphophorus is regulated by the locus P, locus occupied by multiple copies of the melanocortin receptor 4 (MC4R). To demonstrate the involvement of melanocortins in the first sexual maturation, we generated a transgenic zebrafish line ovexpressing agouti-signaling protein (ASIP), an endogenous antagonist that competes with the melanocortin peptides by binding to MCRs. Our results demonstrated that ASIP males reach puberty earlier than wildtype fish. In addition, we demonstrate the presence of MC4R in ovary and testes by in situ hybridization (ISH). In conclusion, we demonstrated that the inhibition of melanocortin signaling by ASIP may modulate to the zebrafish gametogenesis., This work was supported by grants Spanish Science and Education Ministry and European Fund for Regional Development (AGL2013-46448-3-3-R and FEDER). Financial support to Sandra Navarro from Becas Chile 2013-2016

Trabajo presentado en el 11º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Vigo (España), del 13 al 15 de julio de 2017

Trabajo presentado en el 11º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Vigo (España), del 13 al 15 de julio de 2017

Trabajo presentado en el 11º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Vigo (España), del 13 al 15 de julio de 2017, Stress is negatively affecting animal welfare in such a way that most behavioral and physiological functions, such as food intake, are jeopardized. In fish, the stress response initiates with the activation of the hypothalamus-sympathetic-cromaffin (HPC), and the hypothalamus-pituitary-interrenal cells (HPI) axes leading to increased plasma catecholamines and cortisol levels. Our previous results point to the important role played by both dopaminergic and serotonergic systems in initiating the response to acute stress. However, little is known regarding the involvement of both systems under chronic stress and their influence on food intake. To address that question an experiment was performed consisting on stressing rainbow trout (Oncorhynchus mykiss) by high stocking density for 3 and 10 days. Food intake was evaluated all over the experiment in non-stressed and stressed fish. Plasma levels of cortisol, glucose and lactate, the content of DA and 5HT, and their main metabolites (DOPAC and 5HIAA) in different brain regions, and hypothalamic mRNA abundance of TH, TPH1 were assessed. Our results reveal that stressed animals showed reduced food intake, increased plasma cortisol levels, and enhanced dopaminergic and serotonergic activities in telencephalon, hypothalamus, optic tectum and hindbrain, independently of the stress duration. TH and TPH1 mRNA abundance also increased in stressed trout. Our present results support the hypothesis of a key role played by both dopaminergic and serotonergic systems in initiating and maintaining the neuroendocrine response to stress. In addition their involvement in mediating the stress-related food intake inhibition might be taken in consideration., Funded by Ministerio de Economía y Competitividad and European Fund for Regional Development (AGL2013-46448-3-1-R and FEDER) and AEO-ECIMAT. C. Otero-Rodiño was a recipient of FPI fellowship.

Trabajo presentado en el 11º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Vigo (España), del 13 al 15 de julio de 2017

Trabajo presentado en el 11º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Vigo (España), del 13 al 15 de julio de 2017

Trabajo presentado en el 11º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Vigo (España), del 13 al 15 de julio de 2017

Trabajo presentado en el 11º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Vigo (España), del 13 al 15 de julio de 2017, The melanocortin system integrates peptides derived from proopiomelanocortin that show melanocortinic and/or corticotropic activity. Endogenous melanocortin antagonists, agouti-signaling protein (ASIP) and agouti-related protein (AGRP) compete with melanocortin peptide for binding to five different melanocortin receptors (MC1RMC5R). In zebrafish, AGRP and MC5R are duplicated. AGRP1 is specifically expressed in the hypothalamus but AGRP2 is expressed in the pineal. AGPR2 modulates the expression of the melanin-concentrating hormone (MCH) that is involved in the background adaptation. The expression of AGRP2 in the zebrafish pineal gland suggests the involvement of the melanocortin system in the regulation of circadian rhythms. We use a zebrafish transgenic strain overexpressing ASIP (Ag zebrafish) to study the role of melanocortin antagonist in the circadian structure of fish. We demonstrate that both diurnal and nocturnal melatonin levels in Ag zebrafish are statistically similar. Accordingly, comparison of brain transcriptome between Ag and wild type (WT) zebrafish revealed a significant enrichment of tryptophan and circadian rhythms go terms. Finally, the analysis of daily locomotor activity exhibited significant differences between Ag and WT fish further supporting a role for melanocortin system in the activity rhythms of zebrafish.

Trabajo presentado en Aquaculture Europe 19, celebrado en Berlín (Alemania) del 7 al 10 de octubre de 2019., [Introduction]: Feed intake is a critical variable in aquaculture that limits growth and survival of reared animals. The inclusion of new raw materials in fish diets to meet cost-efficient production and sustainability goals may compromise the organoleptic quality of diets and, by extension, fish growth (Yaghoubi et al., 2016). Therefore, it seems reasonable to evaluate the fish s discriminatory capacity towards feed in order to discern the organoleptic preferences of aquacultured species. The goal of this study was to develop an experimental model to test feed discrimination in the gilthead seabream (Sparus aurata), an important species for Mediterranean seawater aquaculture., [Materials and methods]: We set up a dual-choice feeding system using self-feeders activated by a string sensor placed 3cm below the water surface. Feeders were connected to a computer system that recorded the date, time and tank from which each feed demand originated (Leal et al., 2009). The feed reward per sensor activation was set at approximately 1g/demand. Initially, 500 juvenile gilthead sea bream of approximately 10g were maintained in two 2500 l tanks provided with self-feeding systems during eight months for accommodation and learning. Subsequently, animals (body weight around 100g) were transferred to eleven 500 l experimental tanks (n=10/tank) provided with a dual choice feeding system consisting of two string sensors, each activating a different self-feeder. During 28 days, all feeders were provided with a control diet (44% CP and 18% CF, containing 12.5% fishmeal) for accommodation to the experimental tanks, but especially to the dual choice feeding system (phase I). Subsequently (phase II), in four tanks, one feeder was filled up with control diet supplemented with quinine (1.5%, negative diet) whereas the second feeder contained the control diet. In other three tanks, the tester-feeder distributed a positive diet (isoproteic and isolipidic but containing 46% fishmeal, 6% squid meal and 6% krill meal) and the second feeder delivered control diet. Finally, both positive and negative diets were confronted in the remaining four tanks. After eight consecutive days, the position of feeders was switched in the tanks and animals were allowed to feed for further 21 days (phase III). At the end of the experiment, the total amount of feed distributed was calculated by weighing the feed remaining in the feed hoppers. This quantity was used to calculate the delivery rate for each electronic feeder. The amount of feed delivered daily was calculated using the feeder delivery rate and number of daily demands. The experimental tanks were inspected daily to ensure the absence of feed on the bottom. Consecutively, and utilizing the same animals from the previous experiment, an additional trial was set up to corroborate the feeding deterrent effect of quinine using a single feeder per tank for 14 days. Three tanks were fed with quinine-supplemented diet and four tanks with control diet. Feed intake levels were calculated as before., [Results and discussion]: During phase I of the dual-choice experiment, using a univariate general linear model that analyzes all sensors jointly, independently of the tank, no significant differences in feeder activation were found, thus demonstrating that fish demanded feed in a similar way independently of feeder. Sensor activation differed according to time (hour of the day), day and tank. It was demonstrated that fish exhibited a similar daily pattern of demands (figure 1a) in all sensors/feeders. Number of demands was different between tanks but also between the different experimental days, thus indicating the instability of feed intake (data not shown). During phase II, when experimental diets were introduced, we found significant differences in sensor activation, with the lowest values being measured in feeders provided with quinine-supplemented diet and highest demand levels being found in those containing positive diet (figure 1b). This demonstrated that fish exhibited preferences towards sensors coupled to feeders delivering positive diets but avoided feeders supplying a deterrent feed (provided by quinine inclusion), when compared to the control diet. Similarly to the accommodation phase (I), feed intake levels differed according to day and tank, but this time we also recorded significant differences in the 24h-feeding pattern. In this respect, the activation period of sensors delivering quinine-rich diet was narrower (figure 1b). Finally, in phase III when the position of feeders was inverted, fish continued to demand less from feeders delivering the quinine diet, and had higher demands from feeders supplying the positive diet. Results suggest that fish are able to discriminate the position of the feeder according to the type of diet delivered or, what is the same, they can discriminate the diet independently of the feeder position. Similar to phase II, feeder sensor activation differed according to day, tank and time (figure 1c). Finally, using a different experimental set up in which animals were fed exclusively with one type of diet, either control or quinine-supplemented, we confirmed that, when presented with a diet containing quinine, gilthead seabream reduce their voluntary feed intake (data not shown), thus corroborating that the lower preference towards this feed, when an alternative feed is presented, is associated with a feeding deterrent effect., [Conclusions and future directions]: Seabream exhibited variable feed intake levels along different days and fish groups/tanks but were able to discriminate the diet’s organoleptic properties using a dual-choice self-feeding system. This opens the possibility to use the system to evaluate new raw materials in terms of feed acceptance/preference, as well as the potential of flavorings to overcome the negative effects of antinutritional compounds or medication on feed intake.

Congreso de la Asociación Española de Endocrinología Comparada (AIEC), 26-28 Septiembre 2019. Faro, Portugal, In vertebrates, most of the pigment cells derive from the neural crest (NC). The NC is a transient structure of the embryo, which is unique to vertebrate lineage. The development and the evolutionary diversification of neural crest derivatives, the multiple pigment cell types (chromatophores), the types of pigments chromatophores produce, and the different color patterns they generate have been a major contributor to vertebrate phenotypic diversity. To understand the genetic basis of vertebrate chromatophore development and evolution, we reconstructed the evolutionary history of main genetic pathways involved in pigment synthesis and chromatophore development and differentiation. Therefore, using examples from different genetic pathways, I will explore the role of gene and genome duplications as well as gene losses of the pigmentary system and thus its diversification across vertebrate lineages. Our functional genetic studies of pigment cell development in the model (zebrafish and spotted gar) systems of ray-finned fishes in comparison to tetrapods additionally emphasize the great homology and conservation of certain aspects of pigment patterning mechanisms across vertebrates, This work was funded by the Spanish Economy and Competitiveness Ministry projects AGL2011-23581, AGL2014-52473R, AGL2017-89648P to JR. Partial funding was obtained from AGL2016-74857-C3-3-R to JMCR

XVII Congreso Nacional de Acuicultura, Cartagena 7-10 de mayo de 2019