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Temporal signals such as light and temperature cycles profoundly modulate animal physiology and behaviour. Via endogenous timing mechanisms which are regulated by these signals, organisms can anticipate cyclic environmental changes and thereby enhance their fitness. The pineal gland in fish, through the secretion of melatonin, appears to play a critical role in the circadian system, most likely acting as an element of the circadian clock system. An important output of this circadian clock is the locomotor activity circadian rhythm which is adapted to the photoperiod and thus determines whether animals are diurnal or nocturnal. By using a genetically modified zebrafish strain known as Tg (Xla.Eef1a1:Cau.asip1)iim04, which expresses a higher level of the agouti signalling protein 1 (Asip1), an endogenous antagonist of the melanocortin system, we observed a complete disruption of locomotor activity patterns, which correlates with the ablation of the melatonin daily rhythm. Consistent with this, in vitro experiments also demonstrated that Asip1 inhibits melatonin secretion from the zebrafish pineal gland, most likely through the melanocortin receptors expressed in this gland. Asip1 overexpression also disrupted the expression of core clock genes, including per1a and clock1a, thus blunting circadian oscillation. Collectively, these results implicate the melanocortin system as playing an important role in modulating pineal physiology and, therefore, circadian organisation in zebrafish

This paper describes the behavioural tests carried out with real fish to study their behaviour and stress against the different prototypes of mini-robot fish with the aim of identifying stressors and reducing them when designing future robots for aquaculture., Peer Reviewed

Melanocortins are peptides derived from the post-transcriptional processing of proopiomelanocortin precursor (POMC), which encodes several melanocyte-stimulating hormones (MSHs) and adrenocorticotropic hormone (ACTH). Five different receptors (MC1R-MC5R) with discrete functional domains and binding profiles mediate melanocortin signalling. Atypically, the melanocortin system is also regulated by an endogenous antagonist such as agouti-signalling protein (A SIP) and agoutirelated protein (AGRP). The melanocortin system is essential for the regulation of food intake, stress and pigmentation in vertebrates, but only a few studies have focused on its role in behaviour. Here, we review our recent experiments focusing on the involvement of the melanocortin system in the regulation of circadian rhythms using a zebrafish strain overexpressing asip1 ( asip1-Tg). We compared the daily activity rhythms of asip1-Tg and WT strains and found that the circadian activity pattern was completely disrupted in the transgenic animals, which perfectly matched the reduced nocturnal serotonin and melatonin levels in the asip1-Tg animals. The expression of clock genes was also disrupted and the rhythmicity of per1a and clock1a genes was masked by overexpressing asip1. A direct role of ASIP1 in melatonin secretion was confirmed using in vitro pineal culture, which showed a dose-related inhibitory effect on melatonin secretion. Data support that the inhibition of melanocortin system by endogenous antagonists disrupts the central circadian clock masking melatonin secretion and thus the daily activity patterns

Over the last decade, the zebrafish has emerged as an important model organism for behavioural studies and neurological disorders, as well as for the study of metabolic diseases. This makes zebrafish an alternative model for studying the effects of energy disruption and nutritional quality on a wide range of behavioural aspects. Here, we used the zebrafish model to study how obesity induced by overfeeding regulates emotional and cognitive processes. Two groups of fish (n = 24 per group) were fed at 2% (CTRL) and 8% (overfeeding-induced obesity, OIO) for 8 weeks and tested for anxiety-like behaviour using the novel tank diving test (NTDT). Fish were first tested using a short-term memory test (STM) and then trained for four days for a long-term memory test (LTM). At the end of the experiment, fish were euthanised for biometric sampling, total lipid content, and triglyceride analysis. In addition, brains (eight per treatment) were dissected for HPLC determination of monoamines. Overfeeding induced faster growth and obesity, as indicated by increased total lipid content. OIO had no effect on anxiety-like behaviour. Animals were then tested for cognitive function (learning and memory) using the aversive learning test in Zantiks AD units. Results show that both OIO and CTRL animals were able to associate the aversive stimulus with the conditioned stimulus (conditioned learning), but OIO impaired STM regardless of fish sex, revealing the effects of obesity on cognitive processes in zebrafish. Obese fish did not show a deficiency in monoaminergic transmission, as revealed by quantification of total brain levels of dopamine and serotonin and their metabolites. This provides a reliable protocol for assessing the effect of metabolic disease on cognitive and behavioural function, supporting zebrafish as a model for behavioural and cognitive neuroscience.

Feeding motivation plays a crucial role in food intake and growth. It closely depends on hunger and satiation, which are controlled by the melanocortin system. Overexpression of the inverse agonist agouti-signalling protein (ASIP) and agouti-related protein (AGRP) leads to enhanced food intake, linear growth, and weight. In zebrafish, overexpression of Agrp leads to the development of obesity, in contrast to the phenotype observed in transgenic zebrafish that overexpress asip1 under the control of a constitutive promoter (asip1-Tg). Previous studies have demonstrated that asip1-Tg zebrafish exhibit larger sizes but do not become obese. These fish display increased feeding motivation, resulting in a higher feeding rate, yet a higher food ration is not essential in order to grow larger than wild-type (WT) fish. This is most likely attributed to their improved intestinal permeability to amino acids and enhanced locomotor activity. A relationship between high feeding motivation and aggression has been previously reported in some other transgenic species showing enhanced growth. This study aims to elucidate whether the hunger observed in asip1-Tg is linked to aggressive behaviour. Dominance and aggressiveness were quantified using dyadic fights and mirror-stimulus tests, in addition to the analysis of basal cortisol levels. The results indicate that asip1-Tg are less aggressive than WT zebrafish in both dyadic fights and mirror-stimulus tests.

This paper describes the behavioural tests carried out with real fish to study their behaviour and stress against the different prototypes of mini-robot fish with the aim of identifying stressors and reducing them when designing future robots for aquaculture.

This study explored changes in brain serotonin content and activity together with hypothalamic neuropeptide mRNA abundance around feeding time in rainbow trout, as well as the effect of one-day fasting. Groups of trout fed at two (ZT2) and six (ZT6) hours after lights on were sampled from 90 minutes before to 240 minutes after feeding, while additional groups of non-fed trout were also included in the study. Changes in brain amine and metabolite contents were measured in hindbrain, diencephalon and telencephalon, while in the diencephalon the mRNA abundance of tryptophan hydroxylase (tph1, tph2), serotonin receptors (5htr1a, 5htr1b and 5htr2c) and several neuropeptides (npy, agrp1, cartpt, pomca1, crfb) involved in the control of food intake were also assessed. The results showed changes in the hypothalamic neuropeptides that were consistent with the expected role for each in the regulation of food intake in rainbow trout. Serotonergic activity increased rapidly at the time of food intake in the diencephalon and hindbrain and remained high for much of the postprandial period. This increase in serotonin abundance was concomitant with elevated levels of pomca1 mRNA in the diencephalon, suggesting that serotonin might act on brain neuropeptides to promote a satiety profile. Furthermore, serotonin synthesis and neuronal activity appear to increase already before the time of feeding, suggesting additional functions for this amine before and during food intake. Exploration of serotonin receptors in the diencephalon revealed only small changes for gene expression of 5htr1b and 5htr2c receptors during the postprandial phase. Therefore, the results suggest that serotonin may play a relevant role in the regulation of feeding behavior in rainbow trout during periprandial time, but a better understanding of its interaction with brain centers involved in receiving and processing food-related signals is still needed.

In this work, we assessed periprandial serotonin and 5-hydroxyindolacetic acid abundance in several brain areas of rainbow trout, in parallel with the evaluation of gene expression of tryptophan hydroxilase 1 and 2, neuropeptides involved in the central control of food intake, and the quantification of plasma glucose and cortisol. The results showed severe changes in serotonergic systems around mealtime, the most prominent being the increase in their activity just after food intake, suggesting that serotonin has a relevant role in relation to the daily timing of food intake, probably triggering satiety signals. In addition, a temporal adjustment of neuropeptide expression and plasma cortisol was found in relation to food intake, supporting its role in the regulation of feeding behaviour.

[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 investigati

The agouti-signaling protein (ASIP) acts as both a competitive antagonist and inverse agonist of melanocortin receptors which regulate dorsal-ventral pigmentation patterns in fish. However, the potential 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 against infestation and also as a chemical barrier due to its ability to synthesize and secrete mucus and many immune effector proteins. In this study, the putative role of ASIP in regulating the immune system of skin has been explored using a transgenic zebrafish model overexpressing the asip1 gene (ASIPzf). Initially, the structural changes in skin induced by asip1 overexpression were studied, revealing that the ventral skin of ASIPzf was thinner than that of wild type (WT) animals. A moderate hypertrophy of mucous cells was also found in ASIPzf. 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 maintain or 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 yet severe skin injures and a slight increase in mortality was observed. 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.

[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.

In teleost, as in other vertebrates, stress affects reproduction. A key component of the stress response is the pituitary secretion of the adrenocorticotropic hormone (ACTH), which binds to the melanocortin 2 receptor (MC2R) in the adrenal glands and activates cortisol biosynthesis. In zebrafish, Mc2r was identified in male and female gonads, while ACTH has been shown to have a physiological role in modulating reproductive activity. In this study, the hypothesis that other melanocortins may also affect how the zebrafish gonadal function is explored, specifically steroid biosynthesis, given the presence of members of the melanocortin signaling system in zebrafish gonads. Using cell culture, expression analysis, and cellular localization of gene expression, our new observations demonstrated that melanocortin receptors, accessory proteins, antagonists, and agonists are expressed in both the ovary and testis of zebrafish (n = 4 each sex). Moreover, melanocortin peptides modulate both basal and gonadotropin-stimulated steroid release from zebrafish gonads (n = 15 for males and n = 50 for females). In situ hybridization in ovaries (n = 3) of zebrafish showed mc1r and mc4r in follicular cells and adjacent to cortical alveoli in the ooplasm of previtellogenic and vitellogenic oocytes. In zebrafish testes (n = 3), mc4r and mc1r were detected exclusively in germ cells, specifically in spermatogonia and spermatocytes. Our results suggest that melanocortins are, directly or indirectly, involved in the endocrine control of vitellogenesis in females, through modulation of estradiol synthesis via autocrine or paracrine actions in zebrafish ovaries. Adult zebrafish testes were sensitive to low doses of ACTH, eliciting testosterone production, which indicates a potential role of this peptide as a paracrine regulator of testicular function.

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.

Overexpression of asip1 in transgenic zebrafish disrupts dorsoventral pigment pattern in addition to increasing 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. To test this hypothesis, wide scope transcriptomic techniques were used to elucidate the potential pathways involved in the enhanced nutrient absorption and intestinal epithelium permeability/integrity. In addition, the electrogenic capacity for amino acid transport was analysed. Transcriptomic analysis reveal that amino acid, monocarboxylates, ionic and vitamin transmembrane transporters were substantially modified. Enrichment analysis also revealed an inhibition of intestinal lipid metabolism and down-regulation of KEGG pathways related to membrane integrity suggesting augmented intestinal laxity that may enhance paracellular transport. Electrophysiological experiments carried out in Ussing chambers show that asip1 overexpression decrease membraned tissue resistance (Rt), indicating a modification of the intestinal barrier function in ASIP1 transgenic animals. Similarly, paracellular permeability was higher in transgenic zebrafish. 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 also enhanced in transgenic animals providing strong indication that ASIP1 fish can extract more amino acids from their diet at similar feeding levels. Both transcriptomic and electrophysiological results suggest 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. The enhanced growth of ASIP1 zebrafish potentially mediated by improved nutrient uptake and feed efficiency sugges

Self-feeding systems have been extensively used to study animal behaviour and food preferences in vertebrates. In this study, we used a dual-choice self-feeding system to explore feeding behaviour and dietary discrimination based on organoleptic cues. Experimental tanks were provided with 2 self-feeders activated by string sensors and connected to a computer system. In the first experiment (E1) [6 tanks, 10 animals/tank, body weight (BW) 266 g ± 19.4 g], both feeders delivered the same control standard diet to evaluate the potential preference for one feeder. E1 had 3 phases: 1st, animals were allowed to demand from both feeders; 2nd, from only one feeder and 3rd from both feeders again. The second experiment (E2) (11 tanks, 15 animals/tank, BW range 25–50 g) examined whether seabream can discriminate between 3 isoenergetic diets: the control diet; a positive/palatable diet containing high levels of fish, squid and krill meal; and a negative diet in which 1.5% quinine hydrochloride was added to the control diet. E2 had 3 phases: 1st acclimation with control diet; 2nd dual-choice; 3rd dual-choice with reversal of feeders. The third experiment (E3) (8 tanks, 10 animals/tank, BW range 25-50 g) was performed using a single self-feeder per tank, with each tank being fed either control or quinine diet, to corroborate the aversive response to quinine in the absence of dietary choice. E1 demonstrated that animals demanded significantly less when only one feeder/sensor was available. E2 shows that seabream can easily discriminate a bitter compound (quinine) in the diet, showing negative preference (P < 0.05) in both phases, 2nd and 3rd. When no choice was available (E3), seabream equally demanded control or quinine diet, presumably driven by its energy requirements. However, the presence of the bitter substance increased feed refusals, 14.3% rejection of demanded feed in quinine diet, versus 2.6% in control diet, indicating a role of sensory systems in assessing food quality

[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

[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

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

The agouti-signaling protein (ASIP) acts as both a competitive antagonist and inverse agonist of melanocortin receptors which regulate dorsal-ventral pigmentation patterns in fish. However, the potential 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 against infestation and also as a chemical barrier due to its ability to synthesize and secrete mucus and many immune effector proteins. In this study, the putative role of ASIP in regulating the immune system of skin has been explored using a transgenic zebrafish model overexpressing the asip1 gene (ASIPzf). Initially, the structural changes in skin induced by asip1 overexpression were studied, revealing that the ventral skin of ASIPzf was thinner than that of wild type (WT) animals. A moderate hypertrophy of mucous cells was also found in ASIPzf. 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 maintain or 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 yet severe skin injures and a slight increase in mortality was observed. 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

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.

In mammals, glucosensing markers reside in brain areas known to play an important role in the control of food intake. The best characterized glucosensing mechanism is that dependent on glucokinase (GK) whose activation by increased levels of glucose leads in specific hypothalamic neurons to decreased or increased activity, ultimately leading to decreased food intake. In fish, evidence obtained in recent years suggested the presence of GK-like immunoreactive cells in different brain areas related to food intake control. However, it has not been established yet whether or not those neuronal populations having glucosensing capacity are the same that express the neuropeptides involved in the metabolic control of food intake. Therefore, we assessed through dual fluorescent in situ hybridization the possible expression of GK in the melanocortinergic neurons expressing proopiomelanocortin (POMC) or agouti-related protein (AGRP). POMC and AGRP expression localized exclusively in the rostral hypothalamus, in the ventral pole of the lateral tuberal nucleus, the homolog of the mammalian arcuate nucleus. Hypothalamic GK expression confined to the ependymal cells coating the ventral pole of the third ventricle but some expression level occurred in the AGRP neurons. GK expression seems to be absent in the hypothalamic POMC neurons. These results suggest that AGRP neurons might sense glucose directly through a mechanism involving GK. In contrast, POMC neurons would not directly respond to glucose through GK and would require presynaptic inputs to sense glucose. Ependymal cells could play a critical role relying glucose metabolic information to the central circuitry regulating food intake in fish, especially in POMC neurons.

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.

Ghrelin (GRL) is a gut-brain hormone with a role in a wide variety of physiological functions in mammals and fish, which points out the ghrelinergic system as a key element for the appropriate biological functioning of the organism. However, many aspects of the multifunctional nature of GRL remain to be better explored, especially in fish. In this study, we used the CRISPR/Cas9 genome editing technique to generate F0 zebrafish in which the expression of grl is compromised. Then, we employed high-throughput mRNA sequencing (RNA-seq) to explore changes in the brain transcriptome landscape associated with the silencing of grl. The CRISPR/Cas9 technique successfully edited the genome of F0 zebrafish resulting in individuals with considerably lower levels of GRL mRNAs and protein and ghrelin O-acyl transferase (goat) mRNAs in the brain, intestine, and liver compared to wild-type (WT) zebrafish. Analysis of brain transcriptome revealed a total of 1360 differentially expressed genes (DEGs) between the grl knockdown (KD) and WT zebrafish, with 664 up- and 696 downregulated DEGs in the KD group. Functional enrichment analysis revealed that DEGs are highly enriched for terms related to morphogenesis, metabolism (especially of lipids), entrainment of circadian clocks, oxygen transport, apoptosis, and response to stimulus. The present study offers valuable information on the central genes and pathways implicated in functions of GRL, and points out the possible involvement of this peptide in some novel functions in fish, such as apoptosis and oxygen transport.

Melanocortin 4 receptor (MC4R), a canonical melanocyte-stimulating hormone receptor, is the main responsible for monogenic obesity in humans. Previous studies in fish and avian species showed that MC4R becomes an ACTH receptor after interaction with the melanocortin receptor accessory protein 2 (MRAP2). We show that human MC4R behaves in a similar way through its interaction with MRAP2. This evolutionary conservation of MRAP2-induced ligand selectivity supports a physiological role for the interaction with MC4R. Both proteins are coexpressed in the same hypothalamic neurons, providing an anatomical substrate and molecular mechanism for the central therapeutic actions of ACTH in the treatment of infantile spasms. These neurons may link the effects of stress on the energy balance independently of glucocorticoid secretion. The complex MC4R-MRAP2 throws light on the action of ACTH and, by extension, on the relay of stress-related information to additional biological systems.

Serotonin (5-HT) is one of the principal neurotransmitters in the nervous system of vertebrates. It is initially synthesized by hydroxylation of tryptophan (Trp) by means of tryptophan hydroxylase or TPH which is the rate-limiting enzyme in the production of 5-HT. In most vertebrates, there are two isoforms of TPH present, TPH1 and TPH2, which exhibit different catalytic or substrate specificity as well as different expression domains. Studies carried out in mammals show that only tph2 is expressed in the brain whereas tph1-mRNA is primarily localized in the enterochromaffin cells and pineal gland. A large number of neurons are also considered to be serotonergic or “pseudo-serotonergic” as they accumulate and release 5-HT yet do not produce it as no amine-synthetic enzymes are expressed, yet a combination of 5-HT transporters is observed. Therefore, tph expression is considered to be the only specific marker of 5-HT-producing neurons that can discriminate true 5-HT from pseudo-serotonergic neurons. This work examined in situ hybridization to study the mRNA distribution of one paralogue for tph1 and tph2 in the central nervous system of rainbow trout. Results show a segregated expression for both paralogues that predominantly match previous immunocytochemical studies. This study thus adds valuable information to the scarce analyses focusing on the central distribution of the expression of serotonergic markers, particularly tphs, in the vertebrate brain thus characterizing the true serotonergic brain territories.

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 melanocort

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

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

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