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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.

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.

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.

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 el 8th International Symposium on Fish Endocrinology (ISFE), celebrado en Gotemburgo (Suecia), del 26 de junio al 2 de julio de 2016

Proopiomelanocortin (POMC) is a complex precursor that comprises several peptidic hormones, including melanocyte-stimulating hormones (MSHs), adrenocorticotropic hormone (ACTH), and β-endorphin. POMC belongs to the opioid/orphanin gene family, whose precursors include either opioid (YGGF) or the orphanin/nociceptin core sequences (FGGF). This gene family diversified during early tetraploidizations of the vertebrate genome to generate four different precursors: proenkephalin (PENK), prodynorphin (PDYN), and nociceptin/proorphanin (PNOC) as well as POMC, although both PNOC and POMC seem to have arisen due to a local duplication event. POMC underwent complex evolutionary processes, including internal tandem duplications and putative coevolutionary events. Controversial and conflicting hypotheses have emerged concerning the sequenced genomes. In this article, we summarize the different evolutionary hypotheses proposed for POMC evolution.

Comunicación presentada en el 10º Congreso de la Asociación Ibérica de Endocrinología Comparada AIEC, celebrado en Castellón, España, del 23 al 25 de septiembre de 2015

Ponencia presentada en el Congreso Europeo de Acuicultura celebrado en Rodas (Grecia) en octubre de 2011

Comunicación presentada en el XII Congreso Nacional de Acuicultura celebrado en Madrid del 24 al 26 de noviembre de 2009.