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Background: Melatonin is a key hormone in regulation of circadian rhythms, and involved in many rhythmic functions, such as feeding and locomotor activity. Melatonin reportedly counteracts stress responses in many vertebrates, including fish. However, targets for this action of melatonin and underlying mechanisms remain unknown. Results: This study reports potential anti-stress properties of melatonin in goldfish (Carassius auratus), with a focus on its effect on plasma cortisol, food intake, and locomotor activity, all of them involved in the responses to stress exposure. Indeed, acute injection of melatonin counteracted stress-induced hypercortisolinemia and reduced food intake. The reduced locomotor activity following melatonin treatment suggests a possible sedative role in fish. To assess whether this anti-stress effects of melatonin involve direct actions on interrenal tissue, in vitro cultures of head kidney (containing the interrenal cortisol-producing tissue) were carried out in presence of ACTH, melatonin, and luzindole, an antagonist of melatonin receptors. Melatonin in vitro reduced ACTH-stimulated cortisol release, an effect attenuated by luzindole; this suggests the presence of specific melatonin receptors in interrenal tissue. Conclusions: Our data support a role for melatonin as an anti-stress signal in goldfish, and suggest that the interrenal tissue of teleosts may be a plausible target for melatonin action decreasing cortisol production., Ministerio de Ciencia e Innovación (España), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub, Pagado por el autor

The elevation of protein kinase A (PKA) activity activates the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels in urinary bladder smooth muscle (UBSM) cells and consequently attenuates spontaneous phasic contractions of UBSM. However, the role of constitutive PKA activity in UBSM function has not been studied. Here, we tested the hypothesis that constitutive PKA activity is essential for controlling the excitability and contractility of UBSM. We used patch clamp electrophysiology, line-scanning confocal and ratiometric fluorescence microscopy on freshly isolated guinea pig UBSM cells, and isometric tension recordings on freshly isolated UBSM strips. Pharmacological inhibition of the constitutive PKA activity with H-89 or PKI 14-22 significantly reduced the frequency and amplitude of spontaneous transient BK channel currents (TBKCs) in UBSM cells. Confocal and ratiometric fluorescence microscopy studies revealed that inhibition of constitutive PKA activity with H-89 reduced the frequency and amplitude of the localized Ca(2+) sparks but increased global Ca(2+) levels and the magnitude of Ca(2+) oscillations in UBSM cells. H-89 abolished the spontaneous transient membrane hyperpolarizations and depolarized the membrane potential in UBSM cells. Inhibition of PKA with H-89 or KT-5720 also increased the amplitude and muscle force of UBSM spontaneous phasic contractions. This study reveals the novel concept that constitutive PKA activity is essential for controlling localized Ca(2+) signals generated by intracellular Ca(2+) stores and cytosolic Ca(2+) levels. Furthermore, constitutive PKA activity is critical for mediating the spontaneous TBKCs in UBSM cells, where it plays a key role in regulating spontaneous phasic contractions in UBSM., National Institute of Diabetes and Digestive and Kidney Diseases (R01-DK-084284), Depto. de Fisiología, Fac. de Farmacia, TRUE, pub

Obesity is related to vascular dysfunction through inflammation and oxidative stress and it has been identified as a risk factor for chronic renal disease. In the present study, we assessed the specific relationships among reactive oxygen species (ROS), cyclooxygenase2 (COX-2), and endothelial dysfunction in renal interlobar arteries from a genetic model of obesity/insulin resistance, the obese Zucker rats (OZR). Relaxations to acetylcholine(ACh) were significantly reduced in renal arteries from OZR compared to their counterpart, the lean Zucker rat (LZR), suggesting endothelial dysfunction. Blockade of COX with indomethacin and with the selective blocker of COX-2 restored the relaxations to ACh in obese rats. Selective blockade of the TXA2/PGH2 (TP) receptor enhanced ACh relaxations only in OZR, while inhibition of the prostacyclin (PGI2) receptor (IP) enhanced basal tone and inhibited ACh vasodilator responses only in LZR. Basal production of superoxide was increased in arteries of OZR and involved NADPH and xanthine oxidase activation and NOS uncoupling. Under conditions of NOS blockade, ACh induced vasoconstriction and increased ROS generation that were augmented in arteries from OZRandbluntedbyCOX-2 inhibition and by the ROS scavenger tempol. Hydrogen peroxide (H2O2) evoked both endothelium-and vascular smooth muscle (VSM)-dependent contractions, as well as ROS generation that was reduced by COX-2 inhibition.In addition, COX-2 expression was enhanced in both VSM and endothelium of renal arteries from OZR. These results suggest that increased COX-2-dependent vasoconstriction contributes to renal endothelial dysfunction through enhanced (ROS) generation in obesity.COX-2 activity is in turn upregulated by ROS., Ministerio de Economía, Comercio y Empresa (MINECO), Depto. de Fisiología, Depto. de Anatomía y Embriología, Fac. de Farmacia, Fac. de Veterinaria, TRUE, pub

Objetivo: estudiar las distintas formas que la pupila adquiere en las diferentes especies animales. Material y métodos: revisión de la literatura, utilizando PubMed. La estrategia inicial de búsqueda fue pupil shape (limitada a animals). También se revisaron 2 textos relacionados: System of Ophthalmology (Duke-Elder) y Evolution's witness (I. Schwab). Resultados: en los ojos compuestos de los insectos es habitual observar una ilusión óptica que recibe el nombre de pseudopupila. La pupila tiene en la mayor parte de los vertebrados forma circular. Sin embargo en los gatos y en algunas especies de serpiente adopta forma vertical. La apertura vertical podría tener una función fotoprotectora al conseguir una reducción más efectiva de la entrada de luz en el ojo. Se ha especulado con que ayudaría a corregir la aberración cromática, y probablemente ayuda a camuflar al animal. En los rumiantes es habitual la pupila en hendidura horizontal. Esta forma podría potenciar la capacidad del sistema visual para detectar siluetas verticales. En el medio marino es frecuente que la pupila adopte forma de creciente, debido a la presencia de un opérculo superior que protegería la retina del exceso de luz procedente de la superficie. Conclusión: la forma de la pupila ofrece una sorprendente variabilidad gracias a la cual la naturaleza ha adaptado el ojo a diversas circunstancias. Las teorías propuestas para justificar esta gran variabilidad se discuten de forma detallada en el artículo., Objective: To study the different pupil shapes adopted by the different animal species. Material and methods: Review of the related literature, using PubMed database. The initial search strategy was pupil shape (limited to animals). The first volume of System of Ophthalmology (Duke-Elder) and Evolution's witness (I. Schwab) were also reviewed. Results: An optic illusion called pseudopupil is usually observed in the compound eyes of insects. The pupil is circular in most vertebrates, however slit vertical pupils are present in cats and in some snake species. Vertical pupils could have a photoprotective function, as it makes a more complete closure possible in photopic conditions, and helps to camouflage the predator. It has also been hypothesized that it could help to correct chromatic aberration. Ruminants are usually endowed with horizontal pupils. This shape could improve the capacity of the eye to detect vertical silhouettes. Some marine animals have crescent-shaped pupils. In these animals, a superior operculum helps to protect the inferior retina from the great amount of light coming from above. Conclusion: There is a surprising variability in pupil shape. Through this variability, nature has fitted the eye to different circumstances. The theories proposed to explain this high variability are discussed in detail in the article., Depto. de Optometría y Visión, Fac. de Óptica y Optometría, TRUE, pub

Conference of the World-Academy-of-Science-Engineering-and-Technology. Barcelona, SPAIN. OCT 22-24, 2006., We proposed the use of a Toda-Rayleigh ring as a central pattern generator (CPG) for controlling hexapodal robots. We show that the ring composed of six Toda-Rayleigh units coupled to the limb actuators reproduces the most common hexapodal gaits. We provide an electrical circuit implementation of the CPG and test our theoretical results obtaining fixed gaits. Then we propose a method of incorporation of the actuator (motor) dynamics in the CPG. With this approach we close the loop CPG - environment - CPG, thus obtaining a decentralized model for the leg control that does not require higher level intervention to the CPG during locomotion in a nonhomogeneous environments. The gaits generated by the novel CPG are not fixed, but adapt to the current robot bahvior., European Union (SPARK), Universidad Complutense de Madrid, Ministerio de Educación y Ciencia, Ramón y Cajal, Depto. de Análisis Matemático y Matemática Aplicada, Fac. de Ciencias Matemáticas, TRUE, pub

Acute-stress situations in vertebrates induce a series of physiological responses to cope with the event. While common secondary stress responses include increased catabolism and osmoregulatory imbalances, specific processes depend on the taxa. In this sense, these processes are still largely unknown in ancient vertebrates such as marine elasmobranchs. Thus, we challenged the lesser spotted catshark (Scyliorhinus canicula) to 18 min of air exposure, and monitored their recovery after 0, 5, and 24 h. This study describes amino acid turnover in the liver, white muscle, gills, and rectal gland, and plasma parameters related to energy metabolism and osmoregulatory imbalances. Catsharks rely on white muscle amino acid catabolism to face the energy demand imposed by the stressor, producing NH4+. While some plasma ions (K+, Cl− and Ca2+) increased in concentration after 18 min of air exposure, returning to basal values after 5 h of recovery, Na+ increased after just 5 h of recovery, coinciding with a decrease in plasma NH4+. These changes were accompanied by increased activity of a branchial amiloride-sensitive ATPase. Therefore, we hypothesize that this enzyme may be a Na+/H+ exchanger (NHE) related to NH4+ excretion. The action of an omeprazole-sensitive ATPase, putatively associated to a H+/K+-ATPase (HKA), is also affected by these allostatic processes. Some complementary experiments were carried out to delve a little deeper into the possible branchial enzymes sensitive to amiloride, including in vivo and ex vivo approaches, and partial sequencing of a nhe1 in the gills. This study describes the possible presence of an HKA enzyme in the rectal gland, as well as a NHE in the gills, highlighting the importance of understanding the relationship between acute stress and osmoregulation in elasmobranchs., Este trabajo estudia los desequilibrios osmorreguladores y el metabolismo de amino ácidos tras un estrés agudo en la pintarroja (Scyliorhinus canicula). De este modo, se estimula el catabolismo de amino ácidos y la producción del catión amonio (NH4+), que debe ser excretado fuera del cuerpo. Se describen por primera vez transportadores de iones como el intercambiador Na+/H+ (NH4+) sensible a la amilorida en branquias, y la bomba de H+/K+ sensible a omeprazol en glándula rectal. Se describen por tanto efectos fisiológicos al estrés en tiburones que pueden ayudar a mejorar el bienestar de estos animales., Unión Europea, Ministerio de Ciencia e Innovación (MICIN), Ministerio de Agricultura y Pesca, Alimentación y Medio Ambiente (MAPAMA), Agencia Estatal de Investigación. Ministerio de Economía, Industria y Competitividad (MINECO), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The online version of this article contains supplementary material, which is available to authorized users. Received: 11 March 2014 /Accepted: 27 November 2014 /Published online: 15 January 2015, Acid-sensing ion channels (ASICs) are H+-gated, voltage-insensitive cation channels involved in synaptic transmission, mechanosensation and nociception. Different ASICs have been detected in the retina of mammals but it is not known whether they are expressed in adult zebrafish, a commonly used animal model to study the retina in both normal and pathological conditions. We study the expression and distribution of ASIC2 and ASIC4 in the retina of adult zebrafish and its regulation by light using PCR, in situ hybridization, western blot and immunohistochemistry. We detected mRNA encoding zASIC2 and zASIC4.2 but not zASIC4.1. ASIC2, at the mRNA or protein level, was detected in the outer nuclear layer, the outer plexiform layer, the inner plexiform layer, the retinal ganglion cell layer and the optic nerve. ASIC4 was expressed in the photoreceptors layer and to a lesser extent in the retinal ganglion cell layer. Furthermore, the expression of both ASIC2 and ASIC4.2 was down-regulated by light and darkness. These results are the first demonstration that ASIC2 and ASIC4 are expressed in the adult zebrafish retina and suggest that zebrafish could be used as a model organism for studying retinal pathologies involving ASICs., Depto. de Optometría y Visión, Fac. de Óptica y Optometría, TRUE, pub

Calcium balance is of paramount importance for vertebrates. In fish, the endocrine modulators of calcium homeostasis include the stanniocalcin (STC), and some members of the parathyroid hormone (PTH) family, such as the PTH-related protein (PTHrP), acting as antagonists. STC is ubiquitously expressed in higher vertebrates. In turn, bony fish exhibit specific STC-producing glands named the corpuscles of Stannius (CS). Previous studies pointed to a calcium-sensing receptor (CaSR) involvement in the secretion of STC, but little is known of the involvement of other putative regulators. The CS provides a unique model to deepen the study of STC secretion. We developed an ex vivo assay to culture CS of fish and a competitive ELISA method to measure STC concentrations. As expected, STC released from the CS responds to CaSR stimulation by calcium, calcimimetics, and calcilytic drugs. Moreover, we uncover the presence (by PCR) of two PTHrP receptors in the CS, e.g., PTH1R and PTH3R. Thus, ex vivo incubations revealed a dose-response inhibition of STC secretion in response to PTHrP at basal Ca2+ concentrations. This inhibition is achieved through specific and reversible second messenger pathways (transmembrane adenylyl cyclases and phospholipase C), as the use of specific inhibitors highlights. Together, these results provide evidence for endocrine modulation between two antagonist hormones, STC and PTHrP., Foundation for Science and Technology (FST (Portugal), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Phenolic compounds from grape products are known to possess antioxidant activity. Furthermore, grape polyphenols (anthocyanins) could be valorised as natural pigments. However, the dietary use of grape products in laying hen husbandry and its effect on egg quality traits and yolk lipid oxidation have been scarcely studied. In the present research work, egg yolk colour and Haugh units were increased with the intake of grape pomace and extract. Moreover, dietary inclusion of grape pomace at 60 g/kg increased the proportion of polyunsaturated fatty acids in the yolk and improved the yolk lipid oxidative stability during the storage of eggs, whereas no effect was obtained with grape extract. Regarding hen performance, both grape products reduced feed intake, feed conversion ratio and average egg weight, but they did not affect daily egg production and mass. Feeding grape pomace, at 60 g/kg, and grape extract, either at 0.5 or 1.0 g/kg, decreased protein digestibility. In conclusion, the addition of grape products in the diet of laying hens improved the egg quality but reduced feed intake and egg weight. Dietary grape pomace showed higher antioxidant potential in egg yolk than grape extract., Ministerio de Economía, Comercio y Empresa (España), Junta de Castilla y León, Depto. de Genética, Fisiología y Microbiología, Depto. de Producción Animal, Fac. de Ciencias Biológicas, Fac. de Veterinaria, TRUE, pub

The relevance of the insulin-like growth factor-1 (IGF-1) system in several physiological processes is well-known in vertebrates, although little information about their temporal organization is available. This work aims to investigate the possible rhythmicity of the different components of the IGF-1 system (igf-1, the igf1ra and igf1rb receptors and the paralogs of its binding proteins IGFBP1 and IGFBP2) in the liver of goldfish. In addition, we also study the influence of two environmental cues, the light/dark cycle and feeding time, as zeitgebers. The hepatic igf-1 expression showed a significant daily rhythm with the acrophase prior to feeding time, which seems to be strongly dependent on both zeitgebers. Only igfbp1a-b and igfbp1b-b paralogs exhibited a robust daily rhythm of expression in the liver that persists in fish held under constant darkness or randomly fed. The hepatic expression of the two receptor subtypes did not show daily rhythms in any of the experimental conditions. Altogether these results point to the igf-1, igfbp1a-b, and igfbp1b-b as clock-controlled genes, supporting their role as putative rhythmic outputs of the hepatic oscillator, and highlight the relevance of mealtime as an external cue for the 24-h rhythmic expression of the IGF-1 system in fish., Ministerio de Ciencia e Innovación (España), Universidad Complutense de Madrid, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Europe calls for the end to fisheries discards, which means bringing all caught fish (subject to minimum sizes or quotas) to land. This decision is beneficial to the ecosystem, since it forces the selectivity of the fishing gears to improve. However, artisanal fishermen find themselves in a vulnerable situation where their subsistence depends on catches with small profit margins. An exemption to this landing obligation exists, as it is also ruled that those animals whose survival is scientifically guaranteed may be returned to the sea. Here we study the survival of Plectorhinchus mediterraneus captured by hookline and gillnet, as well as their physiological recovery. Survival exceeds 93% in both cases. The physiological assessment of primary (cortisol) and secondary (energy mobilization, acid-base and hydromineral balance, and immune system) stress responses indicates that surviving animals are able to recover after fishing. Thus, we propose the optimal size of capture of this species to achieve greater economic benefit. For this, we rely on the prices according to size in recent years, as well as on the growth curves of the species. In this way, by releasing fish of less than 1 kg, the current benefits could be multiplied between 2.3 and 9.6 times. This pilot study lays the groundwork for regulating artisanal fisheries through scientific data related to survival of discards along with information on the sale prices., Ministerio para la Transición Ecológica y el Reto Demográfico (España), Fundación Biodiversidad, European Commission, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The demographic trend of a species depends on the dynamics of its local populations, which can be compromised by local or by global phenomena. However, the relevance of local and global phenomena has rarely been investigated simultaneously. Here, we tested whether local phenomena compromised a species’ demographic trend using the Eurasian common lizard Zootoca vivipara, the terrestrial reptile exhibiting the widest geographic distribution, as a model species. We analyzed the species’ ancient demographic trend using genetic data from its 6 allopatric genetic clades and tested whether its demographic trend mainly depended on single clades or on global phenomena. Zootoca vivipara’s effective population size increased since 2.3 million years ago and started to increase steeply and continuously from 0.531 million years ago. Population growth rate exhibited 2 maxima, both occurring during global climatic changes and important vegetation changes on the northern hemisphere. Effective population size and growth rate were negatively correlated with global surface temperatures, in line with global parameters driving long-term demographic trends. Zootoca vivipara’s ancient demography was neither driven by a single clade, nor by the 2 clades that colonized huge geographic areas after the last glaciation. The low importance of local phenomena, suggests that the experimentally demonstrated high sensitivity of this species to short-term ecological changes is a response in order to cope with short-term and local changes. This suggests that what affected its long-term demographic trend the most, were not these local changes/responses, but rather the important and prolonged global climatic changes and important vegetation changes on the northern hemisphere, including the opening up of the forest by humans., Ministerio de Economía y Competitividad (MINECO), Ministerio de Educación y Ciencia (MEC) /AEI/ FEDER/ UE, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The use of lysed microalgae in the diet of carnivorous fish can increase the bioavailability of proteins and bioactive compounds, such as unsaturated fatty acids or vitamins in the digestive tract. These are essential molecules for the proper physiological development of fish in aquaculture. However, some antinutritional components and other undesirable molecules can be released from an excess of microalgae supplied, compromising the integrity of the intestine. The inclusion of small amounts of hydrolized microalgae in the fish diet can be a good strategy to avoid negative effects, improving the availability of beneficial compounds. Nannochloropsis gaditana is an interesting microalgae as it contains nutraceuticals. Previous studies reported beneficial effects after its inclusion in the diet of Sparus aurata, a widely cultured species in Europe and in all Mediterranean countries. However, administration of raw microalgae can produce intestinal inflammation, increased intestinal permeability, bacterial translocation and disturbance of digestion and absorption processes. The aim of this study was to evaluate changes in the intestinal microbiota and barrier stability of S. aurata fed with low inclusion (5%) hydrolysed N. gaditana. Intestinal microbiota was analyzed using Illumina MiSeq technology and libraries were constructed using variable regions V3–V4 of 16S rDNA molecules. Analysis were based in the identification, quantification and comparison of sequences. The predictive intestinal microbial functionality was analyzed with PICRUSt software. The results determined that the intestinal microbiota bacterial composition and the predictive intestinal microbiota functionality did not change statistically after the inclusion of N. gaditana on the diet. The study of gene expression showed that genes involved in intestinal permeability and integrity were not altered in fish treated with the experimental diet. The potential functionality and bacterial taxonomic composition, El uso de lisados de microalgas en el cultivo de peces carnívoros puede aumentar la disponibilidad de compuestos bioactivos en el tracto digestivo, mejorando su desarrollo y bienestar. Sin embargo, un exceso de microalgas en la alimentación puede generar algunos componentes antinutricionales, comprometiendo la integridad del intestino. Nannochloropsis gaditana es una microalga interesante, pues contiene compuestos nutracéuticos, aunque su administración en crudo produce inflamación intestinal y cambios en la comunidad bacteriana del intestino, produciendo desequilibrios digestivos y de absorción. En el presente trabajo se han evaluado cambios en la estabilidad y diversidad de la microbiota intestinal de la dorada (Sparus aurata) alimentada con hidrolizados de N. gaditana al 5% de inclusión en el pienso. La microbiota intestinal se analizó mediante secuenciación masiva de ADN ribosómico, mientras que la funcionalidad de la barrera intestinal se evaluó mediante expresión de ciertos genes asociados a esta función. Los resultados muestran que no existen cambios significativos ni en la microbiota ni en la integridad intestinal debidos a la inclusión en la dieta de hidrolizados de N. gaditana al 5 %. Por lo tanto, el presente estudio muestra la posibilidad de complementar la dieta de dorada con hidrolizados de microalgas para mejorar la disponibilidad de compuestos bioactivos., CEIMAR foundation, LifeBioencapsulation S.L, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The aging process can be influenced by environmental factors, such as the social environment. The continuous cohabitation of the chronologically old mice with adult animals improves them at the behavioral level, immune function, oxidative stress and longevity, but causes a deterioration of these parameters in adults. Therefore, the objective of the study was to study whether the coexistence for only 15 min a day of old mice with adult mice, can produce that improvement and greater longevity in old animals without causing deterioration in adults. For that, old and adult CD1 female mice, after two months of that social interaction, were submitted to a behavioral battery and then peritoneal leukocytes were collected to assess several immune functions, oxidative and inflammatory stress parameters as well as catecholamine concentrations. When the adult mice reached old age, the same parameters were again analyzed. The life span of each animal was also recorded. Plasmatic concentration of oxytocin was also studied. The results showed that old mice presented better behavioral capacity, immunity and oxi-inflammatory state after this social interaction with adult animals, and consequently an extended life span. Adult mice, in general, did not show any changes after social interaction with old animals, but when they achieved old age, improvements of some parameters and of longevity were observed in comparison with animals that never had a that social interaction. In conclusion, a short social interaction between old and adult individuals can be an excellent strategy for improving in both the health state and longevity., Universidad Complutense de Madrid, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Vertebrates possess circadian clocks, driven by transcriptional–translational loops of clock genes, to orchestrate anticipatory physiological adaptations to cyclic environmental changes. This work aims to investigate how the absence of a light-dark cycle and a feeding schedule impacts the oscillators in the hypothalamus-pituitary-interrenal axis of goldfish. Fish were maintained under 12L:12D feeding at ZT 2; 12L:12D feeding at random times; and constant darkness feeding at ZT 2. After 30 days, fish were sampled to measure daily variations in plasma cortisol and clock gene expression in the hypothalamus-pituitary-interrenal (HPI) axis. Clock gene rhythms in the HPI were synchronic in the presence of a light-dark cycle but were lost in its absence, while in randomly fed fish, only the interrenal clock was disrupted. The highest cortisol levels were found in the randomly fed group, suggesting that uncertainty of food availability could be as stressful as the absence of a light-dark cycle. Cortisol daily rhythms seem to depend on central clocks, as a disruption in the adrenal clock did not impede rhythmic cortisol release, although it could sensitize the tissue to stress., Ministerio de Ciencia e Innovación (MICINN), Ministerio de Economía Industria y Competitividad (MINECO), Universidad Complutense de Madrid (UCM)/Banco de Santander, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Individuals vary in their immune response and, as a result, some are more susceptible to infectious disease than others. Little is known about the nature of this individual variation in natural populations, or which components of immune pathways are most responsible, but defning this underlying landscape of variation is an essential frst step to understanding the drivers of this variation and, ultimately, predicting the outcome of infection. We describe transcriptome-wide variation in response to a standardised immune challenge in wild feld voles. We fnd that genes (hereafter 'markers') can be categorised into a limited number of types. For the majority of markers, the response of an individual is dependent on its baseline expression level, with signifcant enrichment in this category for conventional immune pathways. Another, moderately sized, category contains markers for which the responses of diferent individuals are also variable but independent of their baseline expression levels. This category lacks any enrichment for conventional immune pathways. We further identify markers which display particularly high individual variability in response, and could be used as markers of immune response in larger studies. Our work shows how a standardised challenge performed on a natural population can reveal the patterns of natural variation in immune response., Natural Environment Research Council (NERC), Depto. de Biodiversidad, Ecología y Evolución, Fac. de Ciencias Biológicas, TRUE, pub

Quantitative PCR (qPCR) has been commonly used to measure gene expression ina number of research contexts, but the measured RNA concentrations do not al-ways represent the concentrations of active proteins which they encode. This canbe due to transcriptional regulation or post-translational modifications, or localiza-tion of immune environments, as can occur during infection. However, in studiesusing free-living non-model species, such as in ecoimmunological research, qPCRmay be the only available option to measure a parameter of interest, and so under-standing the quantitative link between gene expression and associated effectorprotein levels is vital. Here, we use qPCR to measure concentrations of RNA from mesenteric lymphnode (MLN) and spleen tissue, and multiplex ELISA of blood serum to measurecirculating cytokine concentrations in a wild population of a model species, Musmusculus domesticus. Few significant correlations were found between gene expression levels and cir-culating cytokines of the same immune genes or proteins, or related functionalgroups. Where significant correlations were observed, these were most fre-quently within the measured tissue (i.e., the expression levels of genes measuredfrom spleen tissue were more likely to correlate with each other rather than withgenes measured from MLN tissue, or with cytokine concentrations measuredfrom blood). Potential reasons for discrepancies between measures including differences indecay rates and transcriptional regulation networks are discussed. We highlightthe relative usefulness of different measures under different research questionsand consider what might be inferred from immune assays., Biotechnology and Biological Sciences Research Council, Depto. de Biodiversidad, Ecología y Evolución, Fac. de Ciencias Biológicas, TRUE, pub

A new solitary species of Loxosomatidae (Entoprocta), Loxosoma discoides n. sp., is described. The specimens were found in the intertidal level of two locations in the Galician (NW Spain) coast. This new species, with “table-tennis bat” appearance, is characterised by showing large lateral wings in the calyx, which confer the singular aspect. It is the first record of this genus at the coast of the Iberian Peninsula., Depto. de Biodiversidad, Ecología y Evolución, Fac. de Ciencias Biológicas, TRUE, pub

The maintenance of optimal body temperatures has profound consequences on all aspects of ectotherms life history, like fitness and performance, and has been the subject of research for decades. In contrast, for reptiles, comparatively less is known on the potential effects of water balance on physiology, ecology and behaviour. In recent years, several recent studies are pointing out the importance of studying thermoregulation and water balance within the same framework. Here, we used a Mediterranean lizard, Psammodromus algirus, to investigate how field body temperature and water loss rates may vary among populations, between sexes and along an altitudinal gradient. We found little variation in field body temperatures among populations, while within each population, field body temperature may change daily, seasonally and differ between sexes. On the contrary, water loss rates decreased with elevation and showed no seasonal trend or difference between sexes. Microclimatic data indicated that highland lizards experienced more fluctuating conditions, a factor that may explain their lower water loss rates. Other factors, like inter-population differences in ectoparasite intensity, might also contribute in explaining the observed patterns. We present here the first data for a lacertid lizard on the inter-population variability and sexual difference in water loss rates and point out that water balance may play a fundamental role in regulating lizard activity during the hottest and driest period of the year., Ministerio de Economía y Competitividad (MINECO), Fundação para a Ciência e a Tecnologia (FCT, Portugal), Depto. de Biodiversidad, Ecología y Evolución, Fac. de Ciencias Biológicas, TRUE, pub

Typhlocharina is a diverse lineage of minute endogean Anillini (Coleoptera: Carabidae: Trechinae) formed by three genera endemic to the Mediterranean region. Four new species of Microcharidius Coiffait, 1969 from Extremadura (Spain) are described in this work, all of them sharing unusual features of the male genitalia: M. andujari sp.n., M. lencinai sp.n., M. serranoi sp.n., and M. aguiari sp.n. Male genitalia are very important in insect systematics, often bearing diagnostic features involved in prezygotic isolation processes. In Anillini, male genitalia comprise the aedeagus (median lobe and parameres) and ring sclerite, and the aedeagus has been used as a major taxonomic tool to discriminate species. Here we provide an in-depth revision of these structures in Typhlocharina bringing a comparative approach to evaluate their taxonomic potential. The morphological diversity of male genitalia is described in detail, including poorly studied or overlooked characters like ring sclerites, parameres, endophallic sclerites, and apical laminae of the median lobe. The results show phylogenetic patterns and diagnostic differences in male genitalia between Lusotyphlus Pérez-González, Andújar & Zaballos, 2017, Typhlocharis Dieck, 1869, and Microcharidius Coiffait, 1969. Male genitalic anatomy is found to be an efficient taxonomic tool for genus or clade-level recognition, but not for species-specific discrimination, except in a few cases of clear autapomorphies. Also, observed diversity in male genitalia does not have any obvious morphological correlates with the known diversity in female genitalia, but the prevalence of some types of aedeagi in species with unguiform gonocoxites suggests certain parallelisms. The affinities and novel features of the new species are discussed, highlighting the first cases of gradual transition and convergent evolution in acquisition of distinct male genital traits, probably associated with sexual isolation processes. Finally, we use the availab, Universidad Complutense de Madrid (UCM), Depto. de Biodiversidad, Ecología y Evolución, Fac. de Ciencias Biológicas, TRUE, pub

The fungicide vinclozolin (Vz) is an endocrine disruptor with known anti-androgenic activity in vertebrates. However, there is a lack of information about the Vz mode of action in invertebrates, although some studies have shown that this compound can produce alterations in different species. Transcriptional activity was analyzed in the freshwater snail Physella acuta in order to elucidate putative cellular processes altered by this chemical during a response. In order to identify potential molecular biomarkers, a de novo transcriptome was generated for this species that constitutes a valuable source for future studies. This data, together with some already available data, permitted the identification of several genes related to detoxification mechanisms (Cyp2u1, Cyp3a7, Cyp4f22, GSTo1, GSTt2, and MRP1), stress response (Hsp20.4, Hsp17, Hsp16.6, and Cu,Zn-SOD), the hormonal system (Estrogen Receptor and Hsp90), apoptosis (Casp3), and copper homeostasis (ATOX1). Using quantitative Real-Time polymerase chain reaction, mRNA levels of these genes were examined in snails exposed to 20 or 200 µg/L Vz for 24 h. The results showed an overall weak response, with downregulation of Hsp20.4 and no statistically significant change for the other genes. These findings suggest that P. acuta can manage the concentrations of Vz found in the environment with no relevant activation of the pathways analyzed, although additional studies are needed for longer exposure times and including other metabolic pathways. The new genes described open the range of processes that can be studied at the molecular level in toxicity tests., Ministerio de Economía y Competitividad (MINECO), Depto. de Biodiversidad, Ecología y Evolución, Fac. de Ciencias Biológicas, TRUE, pub

The development of frailty scores suitable for mice and which resemble those used in the clinical scenario is of great importance to understand human frailty. The aim of the study was to determine an individual frailty score for each mouse at different ages and analyze the association between the frailty score and its lifespan. For this purpose, the “Valencia Score” for frailty was used. Thus, a longitudinal study in mice was performed analyzing weight loss, running time and speed, grip strength and motor coordination at the late-adult, mature and old ages (40, 56 and 80 weeks old, respectively). These parameters are equivalent to unintentional weight loss, poor endurance, slowness, weakness, and low activity level, respectively, in humans. A cut-off point was used to identify frail mice for each criterion. All the measurements were also performed on chronologically adult prematurely aging mice. The results show that by using the “Valencia Score” for frailty a prematurely aged phenotype can be identified even during the adulthood of animals. This opens up the possibility of carrying out preventive long-term interventions. Moreover, the individual frailty score of a given mouse at the late-adult, mature and old ages is shown to be a relevant predictor of its lifespan., Universidad Complutense de Madrid, Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF), ISCIII/FEDER, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Nocturnin (NOC) is a unique deadenylase with robust rhythmic expression involved in the regulation of metabolic processes in mammals. Currently, the possible presence of NOC in fish is unknown. This report aimed to identify NOC in a fish model, the goldfish (Carassius auratus), and to study the possible regulation of its expression by feeding. Two partial-length cDNAs of 293 and 223 bp, named nocturnin-a (noc-a) and nocturnin-b (noc-b), were identified and found to be highly conserved among vertebrates. Both mRNAs show a similar widespread distribution in central and peripheral tissues, with higher levels detected for noc-a compared with noc-b. The periprandial expression profile revealed that noc-a mRNAs rise sharply after a meal in hypothalamus, intestinal bulb, and liver, whereas almost no changes were observed for noc-b. Food deprivation was found to exert opposite effects on the expression of both NOCs (generally inhibitory for noc-a, and stimulatory for noc-b) in the three mentioned tissues. A single meal after a 48-h food deprivation period reversed (totally or partially) the fasting-induced decreases in noc-a transcripts in all studied tissues and the increases in noc-b expression in the intestinal bulb. Together, this study offers the first report of NOC in fish and shows a high dependence of its expression on feeding and nutritional status. The differential responses to feeding of the two NOCs raise the possibility that they might be underlying different physiological mechanisms (e.g., food intake, lipid mobilization, energy homeostasis) in fish., Ministerio de Economía, Industria y Competitividad (MINECO), Ministerio de Educación y Ciencia (MEC), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Glucose homeostasis is an important biological process that involves a variety of regulatory mechanisms. This study aimed to determine whether ghrelin, a multifunctional gut-brain hormone, modulates intestinal glucose transport in goldfish (Carassius auratus). Three intestinal glucose transporters, the facilitative glucose transporter 2 (GLUT2), and the sodium/glucose co-transporters 1 (SGLT1) and 2 (SGLT2), were studied. Immunostaining of intestinal sections found colocalization of ghrelin and GLUT2 and SGLT2 in mucosal cells. Some cells containing GLUT2, SGLT1 and SGLT2 coexpressed the ghrelin/growth hormone secretagogue receptor 1a (GHS-R1a). Intraperitoneal glucose administration led to a significant increase in serum ghrelin levels, as well as an upregulation of intestinal preproghrelin, ghrelin O-acyltransferase and ghs-r1 expression. In vivo and in vitro ghrelin treatment caused a concentration- and time-dependent modulation (mainly stimulatory) of GLUT2, SGLT1 and SGLT2. These effects were abolished by the GHS-R1a antagonist [D-Lys3]-GHRP-6 and the phospholipase C inhibitor U73122, suggesting that ghrelin actions on glucose transporters are mediated by GHS-R1a via the PLC/PKC signaling pathway. Finally, ghrelin stimulated the translocation of GLUT2 into the plasma membrane of goldfish primary intestinal cells. Overall, data reported here indicate an important role for ghrelin in the modulation of glucoregulatory machinery and glucose homeostasis in fish., Ministerio de Economía y Competitividad (MINECO), Ministerio de Educación y Ciencia (MEC), Natural Sciences and Engineering Research Council (NSERC) of Canada, Saskatchewan Health Research Foundation (SHRF) (Canada), CIHR, Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET) (Argentina), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Alcohol is a serious public health concern that has a differential impact on individuals depending upon age and sex. Patterns of alcohol consumption have recently changed: heavy episodic drinking—known as binge-drinking—has become most popular among the youth. Herein, we aimed to investigate the consequences of intermittent adolescent alcohol consumption in male and female animals. Thus, Wistar rats were given free access to ethanol (20% in drinking water) or tap water for 2-h sessions during 3 days, and for an additional 4-h session on the 4th day; every week during adolescence, from postnatal day (pnd) 28–52. During this period, animals consumed a moderate amount of alcohol despite blood ethanol concentration (BEC) did not achieve binge-drinking levels. No withdrawal signs were observed: no changes were observed regarding anxiety-like responses in the elevated plus-maze or plasma corticosterone levels (pnd 53–54). In the novel object recognition (NOR) test (pnd 63), a significant deficit in recognition memory was observed in both male and female rats. Western Blot analyses resulted in an increase in the expression of synaptophysin in the frontal cortex (FC) of male and female animals, together with a decrease in the expression of the CB2R in the same brain region. In addition, adolescent alcohol induced, exclusively among females, a decrease in several markers of dopaminergic and serotonergic neurotransmission, in which epigenetic mechanisms, i.e., histone acetylation, might be involved. Taken together, further research is still needed to specifically correlate sex-specific brain and behavioral consequences of adolescent alcohol exposure., Instituto de Salud Carlos III (ISC-III) and European Regional Development Funds European Union (ERDF-EU), Ministerio de Economía y Competitividad (MINECO)/ ISC-III, UCM-BSCH, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The importance of the neonatal leptin surge in rodents in neurodevelopmental processes has aroused curiosity in its implication in other physiological systems. Given the role of leptin in neuro-immune interactions, we hypothesized that the neonatal leptin surge could have an effect on the oxidative and inflammatory stress situations of both systems. We blocked the neonatal leptin surge by a leptin antagonist and measured several parameters of oxidative and inflammatory stress in the spleen, hypothalamus and adipose tissue of peripubertal/adolescent rats. The treated rats showed lower activity of several antioxidant enzymes in the spleen and their leukocytes released lower levels of mitogen-stimulated IL-10 and IL-13 and higher levels of TNF-alpha. In conclusion, the neonatal leptin surge may have a key role in the establishment of adequate redox and inflammatory states in the immune system, which is important for the generation of adequate immune responses and to obtain and maintain good health., Ministerio de Ciencia e Innovación (MICINN), Universidad Complutense de Madrid, Redes temáticas de Investigación Cooperativa en Salud (Spain), ISCIII-FEDER of the European Union, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The circadian system is responsible for the temporal organisation of physiological functions which, in part, involves daily cycles of hormonal activity. In this review, we analyse the interplay between the circadian and endocrine systems in fishes. We first describe the current model of fish circadian system organisation and the basis of the molecular clockwork that enables different tissues to act as internal pacemakers. This system consists of a net of central and peripherally located oscillators and can be synchronised by the light–darkness and feeding–fasting cycles. We then focus on two central neuroendocrine transducers (melatonin and orexin) and three peripheral hormones (leptin, ghrelin and cortisol), which are involved in the synchronisation of the circadian system in mammals and/or energy status signalling. We review the role of each of these as overt rhythms (i.e. outputs of the circadian system) and, for the first time, as key internal temporal messengers that act as inputs for other endogenous oscillators. Based on acute changes in clock gene expression, we describe the currently accepted model of endogenous oscillator entrainment by the light–darkness cycle and propose a new model for non-photic (endocrine) entrainment, highlighting the importance of the bidirectional cross-talking between the endocrine and circadian systems in fishes. The flexibility of the fish circadian system combined with the absence of a master clock makes these vertebrates a very attractive model for studying communication among oscillators to drive functionally coordinated outputs., Ministerio de Economía y Competitividad (MINECO), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Mechanical deformability of cells is an important property for their function and development, as well as a useful marker of cell state. The classical technique of micropipette aspiration allows single-cell studies and we provide here a method to measure the two basic mechanical parameters, elastic modulus and Poisson’s ratio. The proposed method, developed from finite-element analysis of micropipette aspiration experiments, may be implemented in future technologies for the automated measurement of mechanical properties of cells, based on the micropipette aspiration technique or on the cell transit through flow constrictions. We applied this method to measure the elastic parameters of lymphocytes, in which the mechanical properties depend on their activation state. Additionally, we discuss in this work the accuracy of previous models to estimate the elastic modulus of cells, in particular the analytical model by Theret et al., widely used in the field. We show the necessity of using an improved model, taking into account the finite size of the cells, to obtain new insights that may remain hidden otherwise., Ministerio de Economía y Competitividad (MINECO), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Ghrelin is an important gut-derived hormone with an appetite stimulatory role, while most of the intestinal hormones, including cholecystokinin (CCK), peptide YY (PYY) and glucagon-like peptide-1 (GLP-1), are appetite-inhibitors. Whether these important peptides with opposing roles on food intake interact to regulate energy balance in fish is currently unknown. The aim of this study was to characterize the putative crosstalk between ghrelin and CCK, PYY and GLP-1 in goldfish (Carassius auratus). We first determined the localization of CCK, PYY and GLP-1 in relation to ghrelin and its main receptor GHS-R1a (growth hormone secretagogue 1a) in the goldfish intestine by immunohistochemistry. Colocalization of ghrelin/GHS-R1a and CCK/PYY/GLP-1 was found primarily in the luminal border of the intestinal mucosa. In an intestinal explant culture, a significant decrease in prepro-cck, prepro-pyy and proglucagon transcript levels was observed after 60 min of incubation with ghrelin, which was abolished by preincubation with the GHS-R1a ghrelin receptor antagonist [D-Lys3]-GHRP-6 (except for proglucagon). The protein expression of PYY and GLP-1 was also downregulated by ghrelin. Finally, intraperitoneal co-administration of CCK, PYY or GLP-1 with ghrelin results in no modification of food intake in goldfish. Overall, results of the present study show for the first time in fish that ghrelin exerts repressive effects on enteric anorexigens. It is likely that these interactions mediate the stimulatory effects of ghrelin on feeding and metabolism in fish., Ministerio de Economía y Competitividad (MINECO), Natural Sciences and Engineering Research Council (NSERC) of Canada, Canada Foundation for Innovation to S. Unniappan, Saskatchewan Health Research Foundation (SHRF), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Purpose: Obesity is associated with impaired immune defences and chronic low levels of inflammation and oxidation. In addition, this condition may lead to premature aging. The aim of the study was to evaluate the effects of a nutritional supplementation with monounsaturated and n-3 polyunsaturated fatty acids on several functions and oxidative stress parameters in peritoneal immune cells of obese mice, as well as on the life span of these animals. Methods: Obesity was induced in adult female ICR/CD1 by the administration of a high-fat diet (HFD) for 14 weeks. During the last 6 weeks of HFD feeding, one group of obese mice received the same HFD, supplemented with 1500 mg of 2-hydroxyoleic acid (2-OHOA) and another with 3000 mg of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Several functions and oxidative stress parameters of peritoneal leukocytes were evaluated. Results: The groups of obese mice treated with 2-OHOA or with EPA and DHA showed a significant improvement in several functions such as chemotaxis, phagocytosis, digestion capacity, Natural killer activity and lymphoproliferation in response to mitogens. All of these functions, which were decreased in obese mice, increased reaching similar levels to those found in non-obese controls. Both treatments also improved oxidative stress parameters such as xanthine oxidase activity, which decreased, catalase activity and glutathione levels, which increased. Conclusion: These data suggest that dietary supplementation with monounsaturated and n-3 polyunsaturated fatty acids could be an effective nutritional intervention to restore the immune response and oxidative stress state, which are impaired in obesemice., Ministerio de Ciencia e Innovación (MICINN), Universidad Complutense de Madrid, Instituto de Salud Carlos III—Fondo Europeo de Desarrollo Regional (ISCIII-FEDER), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Oxidative damage is an important contributor to the morphological and functional changes in osteoporotic bone. Aging increases the levels of reactive oxygen species (ROS) that cause oxidative stress and induce osteoblast apoptosis. ROS modify several signaling responses, including mitogen-activated protein kinase (MAPK) activation, related to cell survival. Both parathyroid hormone (PTH) and its bone counterpart, PTH-related protein (PTHrP), can regulate MAPK activation by modulating MAPK phosphatase-1 (MKP1). Thus, we hypothesized that PTHrP might protect osteoblasts from ROS-induced apoptosis by targeting MKP1. In osteoblastic MC3T3-E1 and MG-63 cells, H2 O2 triggered p38, JNK, ERK and p66Shc phosphorylation, and cell apoptosis. Meanwhile, PTHrP (1-37) rapidly but transiently increased ERK and Akt phosphorylation without affecting p38, JNK, or p66Shc activation. H2 O2 -induced p38 and ERK phosphorylation and apoptosis were both decreased by pre-treatment with specific kinase inhibitors or PTHrP (1-37) in both osteoblastic cell types. These dephosphorylating and prosurvival actions of PTHrP (1-37) were prevented by a phosphatase inhibitor cocktail, the phosphatase MKP1 inhibitor sanguinarine or a MKP1 siRNA. PTHrP (1-37) promptly enhanced MKP1 protein and gene expression and MKP1-dependent catalase activity in osteoblastic cells. Furthermore, exposure to PTHrP (1-37) adsorbed in an implanted hydroxyapatite-based ceramic into a tibial defect in aging rats increased MKP1 and catalase gene expression in the healing bone area. Our findings demonstrate that PTHrP counteracts the pro-apoptotic actions of ROS by a mechanism dependent on MKP1-induced dephosphorylation of MAPKs in osteoblasts., Ministerio de Ciencia e Innovación (MICINN)- Juan de la Cierva (España), Instituto deSalud Carlos III, (España), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Cardiolipin (CL) is a mitochondrial-specific phospholipid. CL content and acyl chain composition are crucial for energy production. Given that estradiol induces CL synthesis in neurons, we aimed to assess CL metabolism in the cerebral cortex (CC) of male and female mice during early postnatal life, when sex steroids induce sex-dimorphic maturation of the brain. Despite the fact that total amount of CL was similar, its fatty acid composition differed between males and females at birth. In males, CL was more mature (lower saturation ratio) and the expression of the enzymes involved in synthetic and remodeling pathways was higher, compared to females. Importantly, the sex differences found in CL metabolism were due to the testosterone peak that male mice experience perinatally. These changes were associated with a higher expression of UCP-2 and its activators in the CC of males. Overall, our results suggest that the perinatal testosterone surge in male mice regulates CL biosynthesis and remodeling in the CC, inducing a sex-dimorphic fatty acid composition. In male’s CC, CL is more susceptible to peroxidation, likely explaining the testosterone-dependent induction of neuroprotective molecules such as UCP-2. These differences may account for the sex-dependent mitochondrial susceptibility after perinatal hypoxia/ischemia., Ministerio de Economía y Competitividad (MINECO), National Agency for Scientific and Technological Promotion (Argentina), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Introducción: La salud depende del buen funcionamiento de los sistemas homeostáticos (el nervioso, endocrino e inmunitario) y de la adecuada comunicación entre ellos. Se ha comprobado que el estado funcional y redox del sistema inmunitario es un excelente marcador de salud, y que una inmunosenescencia prematura supone una menor esperanza de vida. Dado que las catecolaminas modulan la funcionalidad de las células inmunitarias, la alteración en su síntesis podría contribuir a esa inmunosenescencia. Entre las estrategias que se pueden utilizar para controlarla está el ambiente social. Objetivo: Comprobar si una haploinsuficiencia de la tirosina hidroxilasa (TH), enzima limitante de la síntesis de catecolaminas, generaría una inmunosenescencia prematura, y si es posible la modulación de esta por el ambiente social. Material y métodos: Se usaron ratones machos ICR-CD1 adultos (9 ± 1 meses) hemizigotos (HZ) para la tirosina hidroxilasa (TH-HZ) y controles (WT), que fueron distribuidos en cuatro subgrupos: WT > 50% (en la jaula, la proporción de WT fue mayor al 50%), WT < 50%, TH-HZ < 50% y TH-HZ > 50%. En leucocitos peritoneales se valoró la fagocitosis, quimiotaxis y linfoproliferación en presencia de lipopolisacárido. También, la actividad de las enzimas antioxidantes glutatión reductasa y glutatión peroxidasa, y el cociente glutatión oxidado/glutatión reducido. Resultados: Los TH-HZ > 50% presentaron, en leucocitos, una funcionalidad y estado redox deteriorados respecto a WT > 50 y similar a ratones viejos. Sin embargo, los TH-HZ < 50% mostraron valores similares a los WT < 50%. Conclusión: Una haploinsuficiencia de la enzima TH provoca una inmunosenescencia prematura, la cual puede ser compensada por la convivencia con un número apropiado de animales WT., Introduction: Healthy state depends on the appropriate function of the homeostatic systems (nervous, endocrine and immune systems) and the correct communication between them. The functional and redox state of the immune system is an excellent marker of health, and animals with premature immunosenescence show a shorter lifespan. Since catecholamines modulate the function of immune cells, the alteration in their synthesis could provoke immunosenescence. The social environment could be a strategy for modulating this immunosenescence. Aim: To determine if an haploinsufficiency oftyrosine hydroxylase (TH),the limiting enzyme of synthesis of catecholamines, may produce a premature immunosenescence and if this immunosenescence could be modulated by the social environment. Materials and methods: Adult (9 ± 1 months) male ICR-CD1 mice with deletion of a single allele (hemizygotic: HZ) of the tyrosine hydroxylase enzyme (TH-HZ) and wild-type (WT) mice were used. Animals were housed in four subgroups: WT > 50% (in the cage, the proportion of WT mice was higher than 50% in relation to TH-HZ), WT < 50%, TH-HZ < 50% and TH-HZ > 50%. Peritoneal leukocytes were collected and phagocytosis, chemotaxis and proliferation of lymphocytes in the presence of lipopolysaccharide were analyzed. Glutathione reductase and glutathione peroxidase activities as well as oxidized/reduced glutathione ratio were studied. Results: TH-HZ > 50% mice showed a deteriorated function and redox state in leukocytes respect to WT > 50% and similar to old mice. However, TH-HZ < 50% animals had similar values to those found in WT < 50% mice. Conclusion: The haploinsufficiency of TH generates premature immunosenescence, which appears to be compensated by living together with an appropriate number of WT animals., Ministerio de Ciencia e Innovación (MICINN), Instituto de Salud Carlos III (ISCIII), Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidaddel Instituto de Salud Carlos III-FEDER, Universidad Complutense de Madrid, Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas deI ISCIII-FEDER de la Unión Europea, y Grupo de investigación UCM, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Ghrelin, a multifunctional gut-brain hormone, is involved in the regulation of gastric functions in mammals. This study aimed to determine whether ghrelin modulates digestive enzymes in goldfish (Carassius auratus). Immunofluorescence microscopy found colocalization of ghrelin, GHS-R1a and the digestive enzymes sucrase-isomaltase, aminopeptidase A, trypsin and lipoprotein lipase in intestinal and hepatopancreatic cells. In vitro ghrelin treatment in intestinal and hepatopancreas explant culture led to a concentration- and time-dependent modulation (mainly stimulatory) of most of the digestive enzymes tested. The ghrelin-induced upregulations of digestive enzyme expression were all abolished by preincubation with the GHS-R1a ghrelin receptor antagonist [D-Lys3]-GHRP-6, and most of them by the phospholipase C inhibitor U73122 or the protein kinase A inhibitor H89. This indicates that ghrelin effects on digestive enzymes are mediated by GHS-R1a, partly by triggering the PLC/PKC and AC/PKA intracellular signaling pathways. These data suggest a role for ghrelin on digestive processes in fish., Ministerio de Economía y Competitividad (MINECO), Ministerio de Educación y Ciencia (MEC), Natural Sciences and Engineering Research Council (NSERC) (Canada), Canada Foundation for Innovation, Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), (Argentina), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The regulation of food intake in fish is a complex process carried out through several different mechanisms in the central nervous system (CNS) with hypothalamus being the main regulatory center. As in mammals, a complex hypothalamic circuit including two populations of neurons: one co-expressing neuropeptide Y (NPY) and Agouti-related peptide (AgRP) and the second one population co-expressing pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) is involved in the integration of information relating to food intake control. The production and release of these peptides control food intake, and the production results from the integration of information of different nature such as levels of nutrients and hormones as well as circadian signals. The present review summarizes the knowledge and recent findings about the presence and functioning of these mechanisms in fish and their differences vs. the known mammalian model., Ministerio de Ciencia e Innovación (MICINN), Fondo Europeo de Desarrollo Regional (FEDER), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Alcohol use disorder and depression are highly comorbid, and both conditions exhibit important sexual dimorphisms. Here, we aimed to investigate voluntary alcohol consumption after 6 weeks of chronic mild stress (CMS) in Wistar rats – employed as an animal model of depression. Male and female rats were investigated, and changes in several molecular markers were analysed in frontal cortex (FCx) and hippocampal formation (HF). CMS induced depressive-like responses in the forced swimming test - increased immobility time - in male and female animals, without affecting anhedonia (sucrose preference test) nor motor activity (holeboard); body weight gain and food intake were diminished only among CMS males. Voluntary alcohol consumption was evaluated in a two-bottle choice paradigm (ethanol 20% versus tap water) for 4 consecutive days; females exhibited a higher preference for alcohol compared to male animals. In particular, alcohol consumption was significantly higher among CMS females compared to CMS male animals. Remarkably, similar changes in both male and female animals exposed to CMS were observed regarding the expression levels of NCAM-140 KDa (decrease), GFAP and CB1R expression (increase) within the FCx as well as for HF PSD-95 levels (increase). However, contrasting effects in males and females were reported in relation to synaptophysin (SYN) protein levels within the FCx, HF CB1R expression (a decrease among male animals but an increase in females); while the opposite pattern was observed for NCAM-140 KDa protein levels in the HF. A decrease in CB2R expression was only observed in the HF of CMS-females. The present study suggests that male and female animals might be differentially affected by CMS regarding later voluntary alcohol consumption. In this initial approach, cortical SYN, and NCAM-140 KDa, CB1R and CB2R expression within the HF have arisen as potential candidates to explain such sex differences in behaviour. However, the depression-alcoholism relation, Instituto de Salud Carlos III, UCM-BSCH, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Avian embryos are exposed to yolk androgens that are incorporated into the egg by the ovulating female. These steroids can affect several aspects of embryo development, often resulting in increases in overall size or the speed of growth of different traits. However, several studies suggest that they also entail immune costs to the offspring. In this study, we explored whether variation in yolk androgen concentration affected several measures of the constitutive and cell-mediated immune axes in the spotless starling (Sturnus unicolor). Using a within-brood design, we injected different doses of androgens (testosterone and androstenedione) into the eggs. Our study showed that experimentally increased yolk androgens led to sex-specific immunosuppression in both the innate and adaptive axes of the immune system. Both cell-mediated immune response (CMI) and lysozyme activity decreased with increasing androgen levels injected into the egg in the case of male nestlings, whereas there were no effects on females. The effects that we found were always linear: no quadratic or threshold patterns were detected. We found no effects of the experimental treatment in hemolysis or agglutination capacity, but these measures were negatively correlated with CMI, suggesting negative correlation among different branches of the immune system. Blood (trypanosomes and hemosporidians) and intestinal (coccidia) parasites were not affected by the experimental increase of yolk androgen levels. Our results show that in our study species yolk androgens induce immunosuppression in some axes of the male nestling immune system. Further studies should analyze the proximate causes for these contrasting effects in different axes of the immune system and the reason for the differential impact on males and females., Unión Europea. FP7, Ministerio de Ciencia e Innovación (MICINN), Ministerio de Economía y Competitividad (MINECO), Juan de la Cierva Subprogram [MICINN] (Spain), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

New Findings: What is the central question of this study? Evidence is growing for the link between obesity, immune dysfunction and oxidative stress, but it is still not known how the properties and functions of the spleen and splenic leucocytes are affected. What is the main finding and its importance? Obesity led to premature immunosenescence, manifested as oxidative stress and changes in leucocyte functions in mouse spleen. The oleic acid derivative 2-hydroxyoleate and, to a lesser extent, a combination of eicosapentaenoic and docosahexaenoic acids could reverse most of the observed alterations, suggesting a potential therapeutic tool for obesity-related immune dysfunction and redox imbalance. We aimed to investigate the effects of obesity on oxidative stress and leucocyte function in the mouse spleen and to assess whether supplementation with 2-hydroxyoleic acid (2-OHOA) or n-3 polyunsaturated fatty acids (PUFAs) could reverse those effects. Female ICR/CD1 mice (8 weeks old, n = 24) received an obesogenic diet (22% fat for 4 weeks and 60% fat for 14 weeks). After 6 weeks, mice were divided into the following three groups (n = 8 per group): no supplementation; 2-OHOA supplementation (1500 mg kg-1 of diet); and n-3 PUFA supplementation (eicosapentaenoic acid and docosahexaenoic acid, 1500 + 1500 mg kg-1 of diet). Eight mice were fed the standard diet for the whole duration of the study (control group). At the end of the experiment, the following variables were assessed in spleens: levels of reduced (GSH) and oxidized glutathione (GSSG), GSH/GSSG, xanthine oxidase activity, lipid peroxidation, lymphocyte chemotaxis, natural killer activity and mitogen (concanavalin A and lipopolysaccharide)-induced lymphocyte proliferation. Obese animals presented higher GSSG levels (P = 0.003), GSSG/GSH ratio (P = 0.013), lipid peroxidation (P = 0.004), xanthine oxidase activity (P = 0.015) and lymphocyte chemotaxis (P < 0.001), and lower natural killer activity (P = 0.003) and pro, Ministerio de Economía y Competitividad (MINECO), Ministerio de Ciencia e Innovación (MICINN), Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF), Carlos III PRONAOS Study Institute (ISCIII)-Fondo Europeo de Desarrollo Regional (FEDER) of the European Union, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The age-related changes in the immune functions (immunosenescence) may be mediated by an increase of oxidative stress and damage affecting leukocytes. Although the “oxidation-inflammation” theory of aging proposes that phagocytes are the main immune cells contributing to “oxi-inflamm-aging”, this idea has not been corroborated. The aim of this work was to characterize the age-related changes in several parameters of oxidative stress and immune function, as well as in lipofuscin accumulation (“a hallmark of aging”), in both total peritoneal leukocyte population and isolated peritoneal macrophages. Adult, mature, old and long-lived mice (7, 13, 18 and 30 months of age, respectively) were used. The xanthine oxidase (XO) activity-expression, basal levels of superoxide anion and ROS, catalase activity, oxidized (GSSG) and reduced (GSH) glutathione content and lipofuscin levels, as well as both phagocytosis and digestion capacity were evaluated. The results showed an age-related increase of oxidative stress and lipofuscin accumulation in murine peritoneal leukocytes, but especially in macrophages. Macrophages from old mice showed lower antioxidant defenses (catalase activity and GSH levels), higher oxidizing compounds (XO activity/expression and superoxide, ROS and GSSG levels) and lipofuscin levels, together with an impaired macrophage functions, in comparison to adults. In contrast, long-lived mice showed in their peritoneal leukocytes, and especially in macrophages, a well-preserved redox state and maintenance of their immune functions, all which could account for their high longevity. Interestingly, macrophages showed higher XO activity and lipofuscin accumulation than lymphocytes in all the ages analyzed. Our results support that macrophages play a central role in the chronic oxidative stress associated with aging, and the fact that phagocytes are key cells contributing to immunosenescence and “oxi-inflamm-aging”. Moreover, the determination of oxidative stress and i, ISCIII-FEDER of the European Union and Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Ghrelin is the only known hormone posttranslationally modified with an acylation. This modification is crucial for most of ghrelin’s physiological effects and is catalyzed by the polytopic enzyme ghrelin O-acyltransferase (GOAT). The aim of this study was to characterize GOAT in a teleost model, goldfish (Carassius auratus). First, the full-length cDNA sequence was obtained by RT-PCR and rapid amplification of cDNA ends methods. Two highly homologous cDNAs of 1491 and 1413 bp, respectively, named goat-V1 and goat-V2 were identified. Deduced protein sequences (393 and 367 amino acids, respectively) are predicted to present 11 and 9 transmembrane regions, respectively, and both contain two conserved key residues proposed to be involved in catalysis: asparagine 273 and histidine 304. RT-qPCR revealed that both forms of goat mRNAs show a similar widespread tissue distribution, with the highest expression in the gastrointestinal tract and gonads and less but considerable expression in brain, pituitary, liver and adipose tissue. Immunostaining of intestinal sections showed the presence of GOAT immunoreactive cells in the intestinal mucosa, some of which colocalize with ghrelin. Using an in vitro approach, we observed that acylated ghrelin downregulates GOAT gene and protein levels in cultured intestine in a time-dependent manner. Finally, we found a rhythmic oscillation of goat mRNA expression in the hypothalamus, pituitary and intestinal bulb of goldfish fed at midday, but not at midnight. Together, these findings report novel data characterizing GOAT, and offer new information about the ghrelinergic system in fish., Ministerio de Economía y Competitividad (MINECO), Natural Sciences and Engineering Research Council (NSERC) of Canada, Saskatchewan Health Research Foundation (SHRF), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Maternal deprivation (MD) during neonatal life has diverse long-term effects, including modification of metabolism. We have previously reported that MD modifies the metabolic response to high-fat diet (HFD) intake, with this response being different between males and females, while previous studies indicate that in mice with HFD-induced obesity, endocannabinoid (EC) levels are markedly altered in various brown and white adipose tissue depots. Here, we analyzed the effects of MD (24 h at postnatal day 9), alone or in combination with a HFD from weaning until the end of the experiment in Wistar rats of both sexes. Brown and white perirenal and subcutaneous adipose tissues were collected and the levels of anandamide (AEA), 2-arachidonoylglycerol (2-AG), palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) were determined. In males, MD increased the content of OEA in brown and 2-AG in subcutaneous adipose tissues, while in females the content of 2-AG was increased in perirenal fat. Moreover, in females, MD decreased AEA and OEA levels in perirenal and subcutaneous adipose tissues, respectively. HFD decreased the content of 2-AG in brown fat of both sexes and OEA in brown and subcutaneous adipose tissue of control females. In contrast, in subcutaneous fat, HFD increased AEA levels in MD males and OEA levels in control and MD males. The present results show for the first time that MD and HFD induce sex-dependent effects on the main ECs, AEA, and 2-AG, and of AEA-related mediators, OEA and PEA, in the rat brown and white (visceral and subcutaneous) adipose tissues., Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The neonatal leptin surge, occurring from postnatal day (PND) 5 to 13 and peaking at PND9 in rodents, is important for the development of neuroendocrine circuits involved in metabolic control and reproductive function. We previously demonstrated that treatment with a leptin antagonist from PND 5 to 9, coincident with peak leptin levels in the neonatal surge, modified trophic factors and markers of cell turnover and neuronal maturation in the hypothalamus of peri-pubertal rats. The kisspeptin system and metabolic neuropeptide and hormone levels were also modified. Here our aim was to investigate if the timing of pubertal onset is altered by neonatal leptin antagonism and if the previously observed peripubertal modifications in hormones and neuropeptides persist into adulthood and affect male sexual behavior. To this end, male Wistar rats were treated with a pegylated super leptin antagonist (5 mg/kg, s.c.) from PND 5 to 9 and killed at PND102–103. The appearance of external signs of pubertal onset was delayed. Hypothalamic kiss1 mRNA levels were decreased in adult animals, but sexual behavior was not significantly modified. Although there was no effect on body weight or food intake, circulating leptin, insulin and triglyceride levels were increased, while hypothalamic leptin receptor, POMC and AgRP mRNA levels were decreased. In conclusion, alteration of the neonatal leptin surge can modify the timing of pubertal onset and have long-term effects on hypothalamic expression of reproductive and metabolic neuropeptides., Ministerio de Ciencia e Innovación (MICINN), Fondo de Investigación Sanitaria (España), GRUPOS UCM-BSCH, Redes Temáticas de Investigación Cooperativa en Salud (España), Centro de Investigación Biomédica en Red Fisiopatología de Obesidad y Nutrición del Instituto de Salud Carlos III, Fundación de Endocrinología y Nutrición (España), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub