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Background: The aim of this study was to assess the effects that psychological and physiological stressors have on indoor rock climbers, as well as to identify sex differences. Methods: 14 intermediate rock climbers participated in the study, 10 males and 4 females. Mean age was 31 ± 8 years for males and 21 ± 2 years for females. Day 1 consisted of test familiarization and baseline measurements. Day 2 included two test conditions, startle and fatigue, separated by 20 min. In the startle condition, participants had to lead climb a route, and a loud audio stimulus was presented near the top of the climb. In the fatigue condition, participants were required to climb as fast as they could until muscular failure. The competitive state anxiety inventory second review (CSAI-2R) questionnaire was used to assess somatic anxiety, cognitive anxiety, and self-confidence. The four-square step test (FSST) was used to assess motor control, and cortisol levels were acquired via passive drool (PD). Results: Cortisol concentrations were highest in the pre-startle condition (1.72 μg/dL ± 0.66), and values decreased post-startle (1.67 μg/dL ± 0.74) and post-fatigue (1.42 μg/dL ± 0.72). However, cortisol concentrations increased post-startle in females (1.57 μg/dL ± 0.96). Somatic anxiety in males was significantly higher post-startle (16.36 ± 5.54) than pre-startle (14.23 ± 5.09). Females had significantly higher somatic anxiety post-startle (18.00 ± 8.76), and they had lower self-confidence levels (30.00 ± 5.89) than males. Conclusions: There are differences in the way that males and females prepare and respond to stressful situations. Furthermore, time of day may have had a significant impact on cortisol concentrations.

Tools are required for quick and easy preliminary evaluation of functional feeds efficiency on fisheries. The analysis of skin mucus biomarkers is a recent alternative approach providing a faster feed-back from the laboratory which is characterized by being less invasive, more rapid and with reduced costs. The effect of replacing fishmeal and fish protein hydrolysates by means of two porcine by-products, the porcine spray-dried plasma (SDPP) and pig protein hydrolysate (PPH), in compound diets (50.4% crude protein, 16.2% crude protein, 22.1 MJ/kg feed) was evaluated in juvenile meagre (Argyrosomus regius) during a two-months period. To determine the impact of these dietary replacements, growth and food performance were measured together with digestive enzymes activities and filet proximal composition. Additionally, skin mucus was collected and characterized by determining main mucus biomarkers (protein, glucose, lactate, cortisol, and antioxidant capacity) and its antibacterial properties, measured by the quick in vitro co-culture challenges. In comparison to the control group, the inclusion of PPH and SDPP, in meagre diets reduced growth (7.4–8.8% in body weight), increased feed conversion ratios (9.0–10.0%), results that were attributed to a reduction in feed intake values (24.2–33.0%) (P < 0.05). Porcine blood by-products did not modify the activity of gastric and pancreatic digestive enzymes as well as those involved in nutrient absorption (alkaline phosphatase) nor liver oxidative stress condition (P > 0.05). In contrast, a reduction in fillet lipid content associated to an increase in fillet protein levels were found in fish fed SDPP and PPH diets (P < 0.05). As compared to the control diet, the dietary replacement did not alter the levels of the skin mucus biomarkers related to stress (cortisol and antioxidant capacity) or nutritional status (soluble protein, glucose and lactate) (P > 0.05). Interestingly, regardless of the worst performance in somatic growth, meagre fed diets containing both tested porcine by-products showed a significantly improved antibacterial capacity of their skin mucus. This enhancement was more prominent for fish fed with the PPH diet, which may be attributed to a higher content of immunomodulatory bioactive compounds in PPH. Further research will be necessary to provide insights on how the inclusion of SDPP and PPH, at the expense of dietary fishmeal and fish protein hydrolysates, affects feed intake and growth performance in meagre. However, the use of skin mucus biomarkers has been demonstrated to be an excellent methodology for a preliminary characterization of the functional feeds, in particular for their prophylactic properties by the study of mucus antibacterial activity. info:eu-repo/semantics/acceptedVersion

Myogenesis is a complex two-phase process of proliferation and differentiation, which seems to be greatly conserved in vertebrates. For the first time in fish, we identify the changes that occur in the proteome during this process in a gilthead sea bream (Sparus aurata) myocyte primary cell culture (on days 4, 8 and 12), using 2-D gel electrophoresis and LC-MS/MS. A significant increase of myogenin expression at day 8 marked the transition from proliferation to differentiation. Of the 898 spots in the proteome analysis, the 25 protein spots overexpressed on day 4 and the 15 protein spots overexpressed on day 8 indicate the end of proliferation and the beginning of differentiation, respectively. Proliferation was characterized by enrichment of proteins involved in actin cytoskeleton remodelling and in cellular metabolic processes (transcription, ubiquitination, response to stress and glucose metabolism). During differentiation, 41 proteins were overexpressed and 51 underexpressed; many of them related to biosynthetic processes (RNA and protein synthesis and folding, and pentose pathways), terminal myotube formation and muscle contraction. The main cellular processes of both phases of muscle development in fish are similar with those observed in mammals but extended in time, allowing sequential studies of myogenesis.

Fish skin mucus is a viscous and semipermeable barrier made mainly of water, glycoproteins and soluble proteins. It represents an important defence against the environment and previous studies have reported the presence of different substances involved in immune defence responses in it. The aim of the present work was to characterize skin mucus protease activity by zymography and esterase activity of the subfamily of carboxylesterases in three species of interest for aquaculture: gilthead sea bream, sea bass and meagre. Mucus antioxidant power was also determined by adapting ferric reducing antioxidant power (FRAP) analysis. As a result of these non-specific immune defence parameters, we compared the antibacterial capacity of skin mucus in these species via in vitro dual bacteria strains–skin mucus co-culture growths. We used Pseudomonas anguilliseptica and Vibrio anguillarum as marine pathogenic bacteria and Escherichia coli as non-pathogenic. For each fish species, in the respective zymograms, we determined a pattern of proteolytic digestion bands. A high-molecular-weight band (around 200 kDa; H-band) was evident in sea bream and sea bass, and showed chymotrypsin activity. One or two intermediate-molecular-weight bands (around 75 kDa; I-bands) with non-trypsin and non-chymotrypsin activity, and putatively with metalloprotease activity, were evident in all species. Finally, low-molecular-weight bands (between 14 and 30 kDa; L-bands) showed distinct patterns for each species and matched trypsin activity. Despite the conservative pattern of digestion bands, the levels of total proteolytic activity (TPA) were 5 and 10 times higher in meagre than in sea bass and sea bream, respectively. In parallel, three carboxylesterase activities were detected in the mucus of the three fish species, using myristate (pNPM-CE activity), butyrate (pNPB-CE activity) and acetate (pNPA-CE activity) as substrates. Both pNPB-CE and pNPA-CE were the most abundant in fish mucus, and meagre was again the species with the highest levels. In contrast, the antioxidant power of meagre skin mucus was the lowest. We established the capacity of skin mucus to block or limit bacterial growth (lytic activity) using 24 h growth curves. The log-growth phase of V. anguillarum was strongly blocked by sea bream and meagre mucus for a few hours; but not by sea bass mucus. However, if mucus was not renewed, log-growth was at the end of 24 h studied period. For its part, P. anguilliseptica growth curve was delayed by the three mucus types during the entire growth period. Only meagre achieved lytic activity against E. coli growth. All parameters studied here will be of a great interest as non-invasive bioindicators of non-specific immune defences in fish skin mucus.

The European sea bass (Dicentrarchus labrax) is a euryhaline marine teleost that can often be found in brackish and freshwater or even in hypersaline environments. Here, we exposed sea bass juveniles to sustained salinity challenges for 15 days, simulating one hypoosmotic (3‰), one isosmotic (12‰) and one hyperosmotic (50‰) environment, in addition to control (35‰). We analyzed parameters of skin mucus exudation and mucus biomarkers, as a minimally invasive tool, and plasma biomarkers. Additionally, Na+/K+-ATPase activity was measured, as well as the gill mucous cell distribution, type and shape. The volume of exuded mucus increased significantly under all the salinity challenges, increasing by 130% at 50‰ condition. Significantly greater amounts of soluble protein (3.9 ± 0.6 mg at 50‰ vs. 1.1 ± 0.2 mg at 35‰, p &lt, 0.05) and lactate (4.0 ± 1.0 µg at 50‰ vs. 1.2 ± 0.3 µg at 35‰, p &lt, 0.05) were released, with clear energy expenditure. Gill ATPase activity was significantly higher at the extreme salinities, and the gill mucous cell distribution was rearranged, with more acid and neutral mucin mucous cells at 50‰. Skin mucus osmolality suggested an osmoregulatory function as an ion-trap layer in hypoosmotic conditions, retaining osmosis-related ions. Overall, when sea bass cope with different salinities, the hyperosmotic condition (50‰) demanded more energy than the extreme hypoosmotic condition.

The effects of porcine plasma protein hydrolysate (PPH) on growth, feed efficiency, and immune responses was evaluated in Sparus aurata. Fish were fed two isoproteic (48% protein), isolipidic (17% fat), and isoenergetic diets (21.7 MJ/kg) diets, one of them containing 5% PPH at the expense of fishmeal. Both diets were tested for 92 days. A significant increase in growth was observed in fish fed the PPH diet in comparison to the control group (182.2 ± 4.4 vs. 173.8 ± 4.1 g), as well as an increase in feed intake without worsening FCR values. An ex vivo assay, with splenocytes incubated with lipopolysaccharide, was conducted to evaluate the cellular immune competence of fish. Genes involved in humoral immunity (lys, IgM), pro- (tnf-α, il-1β), and anti-inflammatory (tgf-β1, il10) cytokines were upregulated in the PPH group in comparison to the control group. The inclusion of PPH in diets enhanced the antibacterial capacity of skin mucus, as the co-culture of selected bacteria (E. coli, V. anguillarum&nbsp, and P. anguilliseptica) with skin mucus indicated. The present results showed that the PPH in low fishmeal diets (2%) promoted growth and feed efficiency, as well as enhancing the immune response, which indicates that this is a safe and functional ingredient for aquafeeds.

The welfare of fish is influenced by management and environmental factors which may greatly increase animal stress levels and even endanger their survival. In this study of meagre (Argyrosomus regius), two stressor conditions, hypoxia and netting, are employed to evaluate the potential use of fish skin mucus as a non-invasive stress biomarker. Dietary supplementation for a week with stress-attenuating amino acids (aspartate, Asp, 1%; and tryptophan, Trp, 1%) was assayed for both conditions. Mucus and plasma samples were obtained from non-stressed fish (basal), and both one and six post-stress hours; and the levels of glucose, lactate, protein and cortisol were determined. Moreover, the correlations between plasma and mucus stress metabolites and cortisol were established. A classic stress response was evidenced in plasma by increased glucose, lactate and cortisol levels (p

Isotopic signature of fish skin mucus represents an advantageous non-invasive way to study fish ecology and applied biology. Our results demonstrate that stable isotope enrichment in skin mucus is modified by one meal, and the rate of isotope allocation depends on the soluble or insoluble components of the mucus., Fish skin mucus is composed of insoluble components, which form the physical barrier, and soluble components, which are key for interrelationship functions. Mucus is continuously secreted, but rates of production and exudation are still unknown, as are the underlying mechanisms. Using stable isotope analysis, here, we evaluate skin mucus turnover and renewal in gilthead sea bream, separating raw mucus and its soluble and insoluble fractions. Isotopic abundance analysis reveals no differences between mucus and white muscle, thus confirming mucus samples as reliable non-invasive biomarkers. Mucus production was evaluated using a single labelled meal packaged in a gelatine capsule, with both 13C and 15N, via a time-course trial. 13C was gradually allocated to skin mucus fractions over the first 12 h and was significantly (4-fold) higher in the soluble fraction, indicating a higher turnover of soluble mucus components that are continuously produced and supplied. 15N was also gradually allocated to mucus, indicating incorporation of new proteins containing the labelled dietary amino acids, but with no differences between fractions. When existent mucus was removed, dietary stable isotopes revealed stimulated mucus neoformation dependent on the components. All this is novel knowledge concerning skin mucus dynamics and turnover in fish and could offer interesting non-invasive approaches to the use of skin mucus production in ecological or applied biological studies such as climate change effects, human impact, alterations in trophic networks or habitat degradation, especially of wild-captured species or protected species.

10 pages, 5 figures, 1 table supplementary data https://doi.org/10.1016/j.aquatox.2020.105617, In fish, the study of cholinesterases (ChEs) and carboxylesterases (CEs), apart from their involvement in neural activity and xenobiotic metabolism, respectively, requires to be further explored. The European sea bass (Dicentrarchus labrax) was the fish model used to characterise B-esterases in several matrices and organs, as well as to assess the impacts of the insecticide fipronil at two temperatures: the natural temperature at the time of sampling (13 °C) and at 16 °C (based on climate change-related predictions for the Mediterranean region). Fipronil exerts harmful effects in non-target species; however, some countries are reluctant to implement regulations without additional evidence on their toxicity. A comprehensive study was performed in fish pre-acclimated to the two targeted temperatures for 15 days. B-esterases were evaluated in multiple samples after 7 and 14 day exposures to fipronil in feed (dose of 10 mg/kg) and after a 7-day depurative period. Based on hydrolysis rates, results showed that CEs were measurable in all matrices while ChEs were more abundant in muscle and, particularly, acetylcholinesterase (AChE) in the brain. A + 3 °C increase in temperature had little influence on B-esterase activity; however, fipronil caused a significant increase in brain AChE (1.5-fold) and CE (3-fold) activities. Other matrices and organs also experienced alterations in their B-esterase activities that could compromise their physiological functions, This work was financed by the Spanish Ministry of Economy, Industry and Competivity (ref CGL2016-76332-R MINECO/FEDER, UE), With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

Aquaculture has become an agronomic activity with noticeable development around the world to respond to the simultaneous decrease of fish captures and the increasing demand of aquatic products for human consumption. However, different problems limit the development of this industry and one of those is the time required for most of the cultured fish species to achieve economically viable the commercial size. The knowledge up to date of the regulatory systems involved in controlling growth has improved very much but, it is still necessary to devote efforts to transform the basic information in application to fish culture production. The aim of the present review is to summarize the knowledge acquired with the studies about the GH/IGF axis and other hormones regarding their function on the regulation of fish muscle development and growth. To this end, GH and IGFs effects in muscle cells on metabolism and development are examined, as well as the contribution of IGF-I binding proteins, IGF-I receptors and their downstream regulated molecules like TOR and its relation with cell proliferation and differentiation and the myogenic regulatory factors. The effect of regulatory molecules on cultured myocytes are reviewed as well as in vivo responses, including the model of sustained and maintained swimming. Key aspects we consider should be further investigated to complete the scenario of the regulation of fish muscle are also proposed.

It is known that tryptophan (Trp) can attenuate the stress response in fish though its role in chronic stress has been scarcely studied. In this work, the effects of a Trp-enriched diet on stress, metabolism and immune parameters have been analysed from an integrative perspective and using novel methods. Several meagres were submitted to crowding and netting stress during four months. Trp diets did not affect growth or zootechnnical parameters. Plasma stress markers response was not altered significantly in the Trp treatments. Those results were correlated to the mucus parameters and, in addition, several skin immune properties improved in the Trp treatments. Overall, the activity of enzymes related to amino acid and carbohydrate metabolism decreased in the Trp-enriched groups. Trp-supplemented diets increased the liver kynurenine concentration under stress, as well as liver tryptophan in non-stressed fish. The present work shows the first data on the effects of Trp-enriched diets on chronic stress in the meagre. The continuous feeding with 0.25% Trp supplements could improve the meagre welfare, with no effect on productive performance, by keeping or reducing the stress markers; reinforcing the immune system; mitigating the energy metabolism; and enhancing the amino acid reserves.

Low temperatures provoke drastic reductions in gilthead sea bream (Sparus aurata) activity and nourishment, leading to growth arrest and a halt in production. However, scarce data exist concerning the implications of central core control during the cold season. The aim of this work was to study the effects of low temperature and recovery from such exposure on the pituitary activity of sea bream juveniles fed 18% or 14% dietary lipid. A controlled indoor trial was performed to simulate natural temperature fluctuation (22 &deg, C to 14 &deg, C to 22 &deg, C). Meanwhile, we determined the regulatory role of the pituitary by analyzing the gene expression of some pituitary hormones and hormone receptors via qPCR, as well as plasma levels of thyroidal hormones. In response to higher dietary lipids, hormone pituitary expressions were up-regulated. Induced low temperatures and lower ingesta modulated pituitary function up-regulating GH and TSH and thyroid and glucocorticoid receptors. All these findings demonstrate the capacity of the pituitary to recognize both external conditions and to modulate its response accordingly. However, growth, peripheral tissues and metabolism were not linked or connected to pituitary function at low temperatures, which opens an interesting field of study to interpret the hypothalamus&ndash, pituitary&ndash, target axis during temperature fluctuations in fish.

Fish skin mucus is proposed as a novel target for the study of physiological condition and to conduct minimally invasive monitoring of fish. Whereas mucus composition has been a major interest of recent studies, no practical techniques have been proposed to gain understanding of the capacity and rhythm of production and exudation. Here, we used stable isotope analysis (SIA) with a labelled meal, packaged in gelatine capsules, to evaluate mucus production and renewal in a fish model, the gilthead sea bream ( Sparus aurata ). Mucus 13 C- and 15 N-enrichment reached higher levels at 12 h post-ingestion without significant differences at 24 h. When the formation of new mucus was induced, 13 C-enrichment in the new mucus doubled whereas 15 N-enrichment only increased by 10%. These results indicate the feasibility of adopting SIA in mucus studies and allow us to propose this methodology as a means to improve knowledge of mucus turnover in fish and other animals.

Knowledge concerning the health and welfare of fish is important to conserve species diversity. Fish mucosal surfaces, and particularly the skin, are of utmost importance to protect the integrity and homeostasis of the body and to prevent skin infections by pathogens. We performed three trials simulating different environmental and anthropogenic challenges: fish capture (air exposure), bacterial infection and fasting, with the aim of evaluating epidermal mucus as a non-invasive target of studies in fish. In this initial approach, we selected three well-known marine species: meagre (Argyrosomus regius), European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) for our study. Mucus viscosity was measured in order to determine its rheological properties, and mucus metabolite (glucose, lactate, protein and cortisol) levels were analysed to establish their suitability as potential biomarkers. Skin mucus appeared as a viscous fluid exhibiting clearly non-Newtonian behaviour, with its viscosity being dependent on shear rate. The highest viscosity (p

In teleosts, the skin mucus is the first physical barrier against physical and chemical attacks. It contains components related to metabolism, environmental influences and nutritional status. Here, we study mucus and composition based on a proteome map of soluble epidermal mucus proteins obtained by 2D-electrophoresis in gilthead sea bream, Sparus aurata. Over 1300 spots were recorded and the 100 most abundant were further analysed by LC-MS/MS and identified by database retrieval; we also established the related specific biological processes by Gene Ontology enrichment. Sixty-two different proteins were identified and classified in 12 GO-groups and into three main functions: structural, metabolic and protection-related. Several of the proteins can be used as targets to determine fish physiological status: actins and keratins, and especially their catabolic products, in the structural functional group; glycolytic enzymes and ubiquitin/proteasome-related proteins in the metabolic functional group; and heat shock proteins, transferrin and hemopexins, in the protection-related group. This study analyses fish mucus, a potential non-invasive tool for characterising fish status, beyond defence capacities, and we postulate some putative candidates for future studies along similar lines.

There are currently no standard diets or practices to counteract or ameliorate the growth arrest and extremely poor feed conversion rates (FCR) in gilthead sea bream Sparus aurata caused by low water temperatures. The present study analysed the effects of reducing dietary lipid content from 18 to 14% during a temperature fluctuation period. Temperature was gradually reduced from 22° to 14°C over a period of 5 d, and maintained at this low temperature for a further 45 d before gradual recovery over 7 d to 22°C under controlled conditions, avoiding other stressors often present in cage culture conditions. Decreasing dietary lipid content at an elevated temperature (22°C) did not significantly affect the standard growth rate or FCRs of the sea bream, indicating a possibility to reduce the dietary energy during the months before winter. Although cold-induced growth arrest and metabolic depression were unavoidable, a well-formulated, lowlipid diet adequately met the low energy demands at low temperatures. Analysis of plasma metabolites and liver enzyme activities demonstrated a reorganisation of liver metabolism, mainly in fish on a low-lipid diet. Recovery from low temperatures was delayed, with a higher dietary lipid content required for optimal liver condition, and mineral supplementation to maintain osmoregulation and bone formation. Our results provide useful information for fish producers and researchers, suggesting that dietary lipid contents should be reduced to allow fish to better cope with low temperatures, and that improved diets with higher lipid content are required for recovery from low temperatures. © The authors 2018. UID/Multi/04326, PTDC/BIA-ANM/4225/2012 AGL2011-29873 Acknowledgements. This study was supported by a grant from the Spanish government (AGL2011-29873), and by the FTC foundation grants from the Portuguese government (UID/Multi/04326 and PTDC/BIA-ANM/4225/2012). O.T.E. and I.Ö. received fellowships from the Erasmus Turkish Program.

Intensive culture of Senegalese sole, Solea senegalensis, represents an option to diversify Mediterranean aquaculture. As a flatfish species, the fish body presents marked asymmetry between the dorsal or ocular side and the ventral or blind side. Taking into account that different surroundings come into contact with each side, in the present study we aimed to compare the properties of skin mucus from both sides, which is the most external barrier and protection for the fish. Skin mucus has recently been proposed as a novel minimally invasive target for the study of physiological responses. Here, a group of ten Senegalese sole were used to measure basal mucus parameters; then four additional groups of ten specimens each were used to study a hypoxia stress challenge (post-stress groups: 15 min-PS, 1 h-PS, 6 h-PS and 24 h-PS), consisting of a 3-minute air exposure. Physicochemical parameters (viscosity, osmolality, pH and main osmotic ions) were analysed in dorsal and ventral mucus. Thereby, mucus components related to stress responses such as cortisol, glucose, lactate, soluble protein and antioxidant power, were measured and compared between the mucus types and in response to hypoxia. Rheograms revealed similar non-Newtonian behaviour of dorsal and ventral mucus viscosities, which suddenly increased in the 15 min-PS group, thus providing fish with immediate insulating and mechanical protection. However, ventral side viscosity of the 24 h-PS group was lower than basal values and its functionality was compromised. Whereas chemical parameters did not differ between sides or in response to hypoxia, components that are soluble in mucus showed asymmetry with soluble protein and lactate being twofold higher in ventral mucus. The classical stress response of increased plasma cortisol accompanying glucose and lactate release was observed. In parallel, mucus glucose and lactate showed rapid but transitory exudation in the 15 min-PS group, while cortisol was detected at minimal levels in skin mucus, and therefore is not recommended as a mucus indicator for this species. Mucus-soluble protein also rose in the 15 min-PS group, which indicates greater exudation of defensive-related proteins. Finally, the antioxidant power of dorsal mucus increased and accumulated, without returning to basal levels. All these findings indicate that flatfish skin mucus properties depend on animal side, and further studies will be necessary to elucidate whether other mucus defensive parameters show this asymmetry and their relevance in these benthonic species.

Seasonal variations in water temperature are a natural stressor of temperate fish that affect growth performance and metabolism globally. Gilthead sea bream is one of the most economically interesting species in the Mediterranean; but its liver metabolism is affected by the cold season. However, the effects of cold on protein turnover mechanisms have hardly been studied. Here, we study the relationship between liver oxidative status and protein homeostasis pathways during a 50-day low temperature period at 14 °C, and subsequent recovery at two times: 7 days (early recovery) and 30 days (late recovery). Liver redox status was determined by measuring oxidised lipids and proteins, the glutathione redox cycle and major antioxidant enzymes activities. Protein turnover was analysed via liver protein expression of HSP70 and HSP90; proteasome 26S subunits and polyubiquitination, as markers of the ubiquitin–proteasome system (UPS); and cathepsin D, as a lysosomal protease. Low temperature exposure depressed antioxidant enzyme activities, affecting the glutathione redox cycle and reducing total glutathione levels. Both the UPS and lysosomal pathways were also depressed and consequently, oxidised protein accumulated in liver. Interestingly, both protein oxidation and polyubiquitination tagging depended on protein molecular weight. Despite all these alterations, temperature recovery reverted most consequences of the cold at different rates: with delayed recovery of total glutathione levels and oxidised protein degradation with respect to enzyme activities recovery. All these findings demonstrate that protein liver homeostasis is compromised after chronic cold exposure and could be the cause of liver affectations reported in aquaculture of temperate fish.

Ideal nutritional conditions are crucial to sustainable aquaculture due to economic and environmental issues. Here we apply stable isotope analysis as an indicator of fish growth and feeding balance, to define the optimum diet for efficient growing conditions. Juveniles of gilthead sea bream were fed with six isoenergetic diets differing in protein to lipid proportion (from 41/26 to 57/20). As protein intake increased, δ¹⁵N and Δδ¹⁵N of muscle and Δδ¹⁵N and Δδ¹³C of its protein fraction decreased, indicating lower protein turnover and higher protein deposition in muscle. This is reflected in the inverse relationship found between Δδ¹⁵N and growth rate, although no differences were observed in either parameter beyond the protein/lipid proportion 47/23. Principal component analysis (PCA) also signaled 47/23 diet as the pivotal point with the highest growing efficiency, with isotopic parameters having the highest discrimination load. Thus, muscle isotope composition, especially ¹⁵N, can be used to evaluate nutritional status in farmed fish.

Skin and scale formation and regeneration in teleosts have mainly been described from a morphological perspective, and few studies of the underlying molecular events exist. The present study evaluates (1) the change in the skin proteome during its regeneration in a marine teleost fish (gilthead sea bream, Sparus aurata) and (2) the impact of oestradiol-17β (E2) on regeneration and the involvement of oestrogen receptor (ER) isoforms. Thirty-five candidate proteins were differentially expressed (p < 0.05) between intact and regenerated skin proteome 5 days after scale removal, and 27 proteins were differentially expressed after E2 treatment. Agglomerative hierarchical clustering of the skin proteome revealed that the skin treated with E2 clustered most closely to intact skin, while regenerating untreated skin formed an independent cluster. Gene Ontology classification associated the differentially expressed proteins in E2-treated skin with developmental processes and cellular morphogenesis. The proteins modified during skin regeneration suggest a balance exists between immune response and anatomical repair. Overall, the results indicate that, even after 5 days regeneration, the composition of mature skin is not attained, and endocrine factors, in particular E2, can accelerate wound repair acting possibly via ERβs expressed in the skin-scales. Several candidate proteins probably involved in scale development, osteoglycin, lipocalin2 and lamin A and the transcription factors PHD and grainyhead were identified. Future studies of fish skin regeneration will be required to provide further insight into this multistage process, and the present study indicates it will be useful to explore immune adaptations of epithelia permanently exposed to an aqueous environment.

There are few traceability systems other than genetic markers capable of distinguishing between sea products of different origin and quality. Here, we address the potential of stable isotopes combined with metabolic and growth parameters as a discriminatory tool for the selection of fish seeds with high growth capacity. For this purpose, sea bream fingerlings produced in three hatcheries (Spanish Mediterranean coast, MC; Cantabrian coast, CC; and South-Iberian Atlantic coast, AC) were subjected to isotopic analysis (δ15N and δ13C), and indices of growth (RNA and DNA) and energy metabolism [cytochrome-c-oxidase (COX) and citrate synthase (CS) activities] were calculated. These analyses were performed prior to and after a "homogenization" period of 35 days under identical rearing conditions. After this period, fingerlings were discriminated between hatcheries, with isotopic measures (especially δ15N), metabolic parameters (COX and CS), and proximal composition (fat content) in muscle providing the highest discriminatory capacity. Therefore, particular rearing conditions and/or genetic divergence between hatcheries, affecting the growth capacity of fingerlings, are defined mainly by the isotopic imprint. Moreover, the muscle isotopic signature is a more suitable indicator than whole fish for discrimination purposes.

Here we examined the use of stable isotopes, [13C]starch and [15N]protein, as dietary tracers to study carbohydrate assimilation and distribution and protein utilisation, respectively, by rainbow trout (Oncorhynchus mykiss). The capacity of glucose uptake and use by tissues was studied, first, by varying the digestibility of carbohydrate-rich diets (30 % carbohydrate), using raw starch and gelatinised starch (GS) and, second, by observing the effects of two regimens of activity (voluntary swimming, control; sustained swimming at 1·3 body lengths/s, exercise) on the GS diet. Isotopic ratio enrichment (13C and15N) of the various tissue components (protein, lipid and glycogen) was measured in the liver, muscles, viscera and the rest of the fish at 11 and 24 h after a forced meal. A level of 30 % of digestible carbohydrates in the food exceeded the capacity of rainbow trout to use this nutrient, causing long-lasting hyperglycaemia that raises glucose uptake by tissues, and the synthesis of glycogen and lipid in liver. Total13C recovered 24 h post-feeding in the GS group was lower than at 11 h, indicating a proportional increase in glucose oxidation, although the deposition of lipids in white muscle (WM) increased. Prolonged hyperglycaemia was prevented by exercise, since sustained swimming enhances the use of dietary carbohydrates, mainly through conversion to lipids in liver and oxidation in muscles, especially in red muscle (RM). Higher recoveries of total15N for exercised fish at 24 h, mainly into the protein fraction of both RM and WM, provide evidence that sustained swimming improves protein deposition, resulting in an enhancement of the protein-sparing effect.

The influence of a daily oral dose of alpha-ketoglutarate (AKG, 0.1 g/kg body weight), an intermediate metabolite in the Krebs cycle and a dietary additive, on the pituitary proteome of gilthead sea bream was determined by two-dimensional electrophoresis (2-DE). A high-resolution map of the sea bream pituitary proteome was generated. Proteins with a modified expression between Controls and AKG treated fish were further analysed by MALDI-TOF/TOF-MS and liquid chromatography combined with a nanoelectrospray (LC–MS/MS). The main changes in the proteome induced by AKG treatment were grouped. Metabolic proteins up-regulated with AKG supplementation included fructose-bis-phosphate aldolase, glyceraldehyde-phosphate dehydrogenase and malate dehydrogenase, all related to glucose metabolism (p < 0.000). Protein folding related up-regulation with AKG supplementation included two isoforms of heat shock proteins as well as cyclophylin and chaperonin (p < 0.000). An unexpected form of apolipoprotein- A-1 with lower molecular weight (15–16 kDa) was evidenced as being highly abundant in the pituitary proteome of Controls, yet it was down-regulated by AKG treatment. Finally, proteins found to be associated with regeneration of neural function namely cofilin and Vat-protein were up-regulated after AKG supplementation. The only hormone to be modified by AKG treatment was somatolactin, which was significantly down-regulated cf. Controls. In summary, these results provide evidence of a potential endocrine/metabolic regulatory loop activated by AKG supplementation. This research was funded by an AQUAGENOME (SSP8, No. 044481) resource exchange grant and pluriannual funding to CCMAR from the Portuguese Science and Technology Foundation (FCT) and XRAq projects (Generalitat de Catalunya, Spain). A.I. was recipient of a research grant from the Spanish Government (Programa de Ayudas de Mobilidad José Castillejo).

Two groups of juvenile gilthead sea bream were kept on two different swimming regimes (Exercise, E: 1.5 body length s(-1) or Control, C: voluntary activity) for 1 month. All fish were first adapted to an experimental diet low in protein and rich in digestible carbohydrates (37.2% protein, 40.4% carbohydrates, 12.5% lipid). The cellularity and capillarisation of white muscle from two selected areas (cranial (Cr), below the dorsal fin, and caudal (Ca), behind the anal fin) were compared. The body weight and specific growth rate (SGR) of group E rose significantly without an increment in feed intake, pointing to higher nutrient-use efficiency. The white muscle fibre cross-sectional area and the perimeter of cranial samples increased after sustained activity, evidencing that sustained exercise enhances hypertrophic muscle development. However, we cannot conclude or rule out the possibility of fibre recruitment because the experimental period was too short. In the control group, capillarisation, which is extremely low in gilthead sea bream white muscle, showed a significantly higher number of fibres with no surrounding capillaries (F0) in the cranial area than in the caudal area, unlike the exercise group. Sustained swimming improved muscle machinery even in tissue normally associated with short bouts of very rapid anaerobic activity. So, through its effect on the use of tissue reserves and nutrients, exercise contributes to improvements in fish growth what can contribute to reducing nitrogen losses.

During winter, low temperatures induce a direct metabolic depression in gilthead sea bream, without any significant compensatory effect below 13 °C. The present study therefore focused on how to improve response to cold in these fish, looking specifically at the two factors of diet (high energy, HiE, and low energy, LoE) and activity (normal, − SW, and sustained activity, + SW) prior to exposure to cold. Following a preparatory period of 75 days water was adjusted to 10 °C and kept for 40 days. Enzymatic activities and store deposition revealed that the HiE−SW group had acquired an energy surplus whilst the LoE+SW group exhibited an energy deficit. Liver enzyme activities evidenced diet dependence: LoE groups showed greater glucose-6-phosphate dehydrogenase activity and HiE groups showed greater lipoprotein lipase and hepatic lipase activities. Moreover, the HiE−SW group's lower citrate synthase/cytochrome- c -oxidase ratio reflected the energy surplus available. Perivisceral fat mobilisation caused by cold stress affected liver integrity, resulting in a pre-steatotic condition for the HE−SW group. The differences in liver enzyme activities produced by pre-cold conditions disappeared at low temperatures and enzymatic activities did not compensate. Therefore any improvement that would enable gilthead sea bream to face up to winter must be achieved prior to the appearance of low temperatures.

Although gilthead sea bream have been cultured successfully for the last two decades they are particularly sensitive to low temperature. Especially in the northern Mediterranean area, cold affects fish health and decreases fish-farm production, and may even cause mortality through what is known as ‘Winter Disease’ or ‘Winter Syndrome’. This paper reviews the diagnosis and physiological effects of this disease, focusing on recent studies of cold-induced alterations in gilthead sea bream physiology. ‘Winter Syndrome’ is characterised by multi-organ dysfunction entailing hyposensitivity, erratic swimming, pale and friable livers, necrotic muscles, atrophy of the exocrine pancreas, and distended digestive tract. Its complex aetiology involves several factors such as thermal stress, metabolic depression, immune suppression, and occasional opportunistic pathogens. Low temperatures may be the initial cause of all these factors, except pathogen action. Indoor studies have demonstrated that a drop in temperature causes cold-induced fasting, thermal stress and metabolic depression. These immediate effects are related to an ionic imbalance caused by malfunctions of the gills and digestive system. They are also related to a fatty liver, which appeared steatotic and affected hepatic metabolism and blood composition. The result is a lower immune capacity and fish that are more susceptible to infection. There is no significant thermal compensation under cold conditions and in this situation any additional stress factors can cause fish to suffer metabolic collapse. This study reviews the physiological and zootechnical origins of the disease and, where possible, recommends ways of improving culture conditions during pre-cold, cold and recovery periods.

Gilthead sea bream exposed to the cold show multiple physiological alterations, particularly in liver. A typical cold-stress response was reproduced in gilthead sea bream acclimated to 20 degrees C (Warm group) when the water temperature was lowered to 8 degrees C (Cold group). After 10 days, thiobarbituric acid reactive substances in the liver had increased by 50%, and nitric oxide had increased twofold. This indicates that lipid peroxidation and oxidative stress had occurred. Protein profiles of liver from fish in warm and cold environments were obtained by 2-DE. Quantification of differential expression by matching spots showed that a total of 57 proteins were altered significantly. Many proteins were downregulated following cold exposure, including actin, the most abundant protein in the proteome; enzymes of amino acid metabolism; and enzymes with antioxidant capacity, such as betaine-homocysteine-methyl transferase, glutathione-S-transferase and catalase. Some proteins associated with protective action were upregulated at low temperatures, including peroxiredoxin, thioredoxin and lysozyme; as well as enzymes such as aldehyde dehydrogenase and adenosin-methionine synthetase. However, the upregulation of proteases, proteasome activator protein and trypsinogen-like protein indicated an increase in proteolysis. Increases in elongation factor-1alpha, the GAPDH oxidative form, tubulin and Raf-kinase inhibitor protein indicated oxidative stress and the induction of apoptosis. These data indicate that cold exposure induced oxidative damage in hepatocytes.

To differentiate between cold and fasting effects on gilthead sea bream (Sparus aurata) physiology, three conditions (cold-induced fasting at 8 °C, cold group; forced fasting at 18 °C, fasting group, and cyclic thermal fluctuations between 16 °C and 8 °C, fluctuating group) were compared with a control group (feeding at 18 °C). Oxygen consumption rate, plasma lipid fractions, and tissue composition were analysed either during long exposure (5 weeks) or after 20 days of recovery. Sea bream showed strict thermoconformism in metabolic rate and feeding, and all groups, after changes, mobilised perivisceral fat. The fasting group also consumed other tissue reserves sequentially, except nonpolar lipids in liver. In the cold group, this liver lipid fraction increased twofold, but in the fluctuating group, food intake was gradually restored during each short period of higher temperature, and the steatotic livers caused by cold were not seen. During recovery, the fasting group showed the highest growth rate and deposition of reserves, whereas the cold group, although they mobilised liver lipid, did not grow. Appropriate management of the transient short periods of higher temperature may improve recovery from the cold season in gilthead sea bream culture.

Gilthead sea bream cultured along the northern Mediterranean coast are affected by the winter season when low temperatures reduce fish feed intake and growth. The coldest episodes can provoke a fish pathology known as ‘winter disease’. The effects of low temperatures, as well as concurrent fasting, have been studied by transferring three groups of gilthead sea bream from 16 °C to 14 °C, 12 °C and 8 °C. Fish at 12 °C and 8 °C refused food, whereas those at 14 °C were not fed following the temperature drop. Changes in body indices, organ composition, liver metabolism, and in particular, lipid fractions and their fatty acids were analysed on days 7 and 20 after the temperature shift. Only the rapid reduction of non-polar lipids in muscle was common for the three conditions. Fasting effects were linked to the maintenance temperature, being maximal after 20 days at 14 °C where fish body weight, hepatosomatic index, and perivisceral fat were reduced by 18%, 40%, and 60%, respectively. In this group, liver lipids did not change, as was the case for the enzymatic activities of liver glucose-6-phosphate and phosphogluconic acid dehydrogenases (G6PDH and PGADH) and lipoprotein lipase (LPL). In contrast, the liver of sea bream submitted to 8 °C accumulated large amounts of non-polar lipids (from 80 mg to 125 mg in 20 days), changing in size and aspect (bigger, pale, and friable). Simultaneously, liver LPL and hepatic lipase (HL) activities decreased. After 20 days at 8 °C, sea bream exhibited incipient acclimation responses to low temperatures: rising levels of unsaturation ratio in gill and liver polar lipids, of docosahexaenoic acid (DHA) in muscle polar lipids, and of G6PDH and PGADH hepatic activities. Fish at 12 °C presented some changes similar to those of the group at 14 °C (e.g., in morphological indices, and LPL and HL activities) and others like the group at 8 °C (increases in G6PDH and PGADH), suggesting a temperature threshold for gilthead sea bream (below 13 °C).

Cultured gilthead sea bream (Sparus aurata L.) are exposed to a multifactorial disease termed “winter syndrome”, and low temperatures are the recurrent inducing factor. The effects of low temperatures on tissue composition and fatty acid profiles of polar and non-polar lipid fractions of sea bream were studied in two conditions: a gradual temperature drop (GD: from 18 °C to 8 °C at a rate of 1 °C·day− 1) or two sharp drops (SD: from 18 °C to 12 °C and then from 12 °C to 8 °C completed within a day, with a 1 week interval). No significant differences were detected between GD and SD in any of the variables studied. Animals stopped feeding when the temperature fell below 13 °C, and the resulting fast provoked body weight losses of 12% (GD) and 10% (SD) at the end of the experiment (15th day), and decreased non-polar lipids content in muscle. Unsaturation of polar fatty acids in muscle (SD) and in gills (SD and GD) rose, demonstrating a homeoviscous acclimation of membranes to cold. In contrast, unsaturation of liver polar fatty acids did not rise, but liver showed a great increase in total lipid content on day 15 (+ 35% SD, and + 43% GD), caused by the increase in non-polar lipids, especially n-3 fatty acids. The high deposition of these essential fatty acids, mainly 22:6n-3, in fasting sea bream implied mobilization from extra-hepatic stores and caused dramatic modifications in the physical properties of the liver (larger, more friable and yellowish), often associated with winter disease. Moreover, plasma aspartate aminotransferase (ASAT) levels rose three-fold in the two conditions. No fish succumbed to winter disease, but the incipient appearance of symptoms was evident. The relationships between the changes in muscle, gill, and, especially liver, at low temperature and the clinical signs of ‘winter disease’ are discussed.

Low temperature has been implicated in inducing outbreaks of ‘winter syndrome’ or ‘winter disease’ in farmed gilthead sea bream (Sparus aurata). The responses of gilthead sea bream to reduced temperature followed by maintenance at low temperaturewere studied. In a first experiment, oxygen consumptionwas measured when water temperature was reduced from 18°C to 8 °C at either a rate of 1 °C· day-1 or as two ‘sharp drops’ (from 18 °C to 12 °C, and from 12 °C to 8 °C). In a second experiment, the water temperature was reduced from 16 °C to 8 °C or 12 °C and then maintained for 20 days to study the fish acclimation to these temperatures. In both experiments, fish stopped feeding below 13 °C and did not resume feeding when maintained at low temperatures. The decrease in metabolic activity, expressed by the oxygen consumption rate, was directly related to the fall in water temperature: the Q10(18 °C-8 °C) values were between 2.2–2.5, independently of the descend rate in water temperature. However, we observed a more reduced metabolic rate when the water temperature was below 12 °C. Fish maintained at low temperatures showed only a partial recovery in oxygen consumption (15% at 8 °C and 20% at 12 °C) after 20 days. A higher metabolic rate together with a fasting-temperature condition meant that maintenance at 12 °C was more aggressive than at 8 °C, as revealed by the condition factor and energy needs. Data suggest that 12 .C could be a threshold temperature for the metabolic activity of gilthead sea bream. The relationship between low temperatures and their possible implication in the appearance of ‘winter disease’ in gilthead sea bream is also discussed.

Gilthead sea bream obtained from a fish farm in the Delta de l'Ebre (Tarragona, Spain) were used to study various functional alterations associated with “winter syndrome.” The fish groups considered were: control fish routinely sampled 3 weeks before an outbreak (which took place in March 1998); winter syndrome symptomatic fish (assessed by eye and corroborated through histopathological analysis); and asymptomatic animals sampled from the same tank where winter syndrome fish were found. Winter syndrome animals weighed less than control and asymptomatic fish. The symptomatic animals suffered a drop in the haematocrit and number of erythrocytes, although red cell volume did not change. Furthermore, the concentration of total plasma proteins in winter syndrome fish was higher than in control animals, due to a rise in β2- and γ-globulins; the α2-globulin concentration was lower and albumin and α1- and β1-globulins remained unchanged. A significant increase in the concentration of plasma amino acids, both essential and nonessential, was found in symptomatic fish. This group of animals showed a lower plasma glucose concentration. There were no differences in either the plasma sodium or chloride concentrations between control and symptomatic fish. Instead, the plasma potassium concentration in winter syndrome animals doubled that of control animals. The plasma concentrations of calcium and magnesium were lower in symptomatic animals than in control fish. Symptomatic fish showed high lipid concentration in liver and elevated plasmatic GOT activity, indicating hepatic damage. No other significant differences in the composition of liver and white muscle were found between control and symptomatic animals.

Low temperatures, and the voluntary fasting that they induce, have been implicated in outbreaks of winter syndrome in farmed gilthead sea bream (Sparus aurata). Two experiments were performed with the objective of studying if a decrease in water temperature is sufficient to induce this disease or some of its associated signs. In the first experiment, water temperature was either acutely or gradually reduced to 8 °C. In the second experiment, fish were exposed to either 8 °C or 12 °C, or fasted at 14 °C. Although no mortalities due to winter syndrome were registered during any of the experiments, some of the signs described in affected sea bream were observed in 8 °C-exposed fish. Among the most relevant were the paleness and friability of the liver and the occurrence of fatty degeneration in the hepatocytes. During the experiments, the general state of health of fish was monitored by measuring hematic parameters and the plasma concentration of proteins, glucose and ions. Low temperatures (8 and 12 °C) and fasting at 14 °C resulted in significant decreases in the levels of the different plasma protein fractions. This drop was more important in all the 8 °C-exposed fish, due mainly to a higher descent of albumin, α1-globulins and fibrinogen. Moreover, 8 °C-exposed fish showed a fall in total white blood cells and a rise of plasma glycemia, as well as a significant drop of plasma potassium and calcium levels and a transient increase of plasma magnesium concentration. In 8 °C-exposed fish, the rate of water temperature descent did not modify the profiles of change of any of the studied parameters. These results indicate that at 8 °C gilthead sea bream are unable to maintain the levels of plasma protein fractions and ions present in control animals.

The osmoregulation capabilities of juvenile Siberian sturgeon exposed to three experimental osmolalities (22, 250 and 387 mOsmol kg−1) were studied over a 45-day period. Growth performance, haematological parameters, ion concentrations, gill and spiral valve Na+-K+-ATPase activities, as well as gill and spiral valve histology, were measured. At the end of the period, the plasma osmolality of fish kept in 250 and 387 mOsmol kg−1 was higher than that of fish kept in 22 mOsmol kg−1. Similar trends were observed in electrolyte concentrations. Spiral valve and gill Na+-K+-ATPase activity varied with exposure time and environmental salinity. Shortly after being transferred to hyperosmotic media, spiral valve Na+-K+-ATPase activity fell, while gill Na+-K+-ATPase activity remained constant. At the end of the experiment, gill Na+-K+-ATPase activities in fish kept in isosmotic and hyperosmotic media had increased in comparison to those of the control fish. Moreover, spiral valve Na+-K+-ATPase activities recovered and were similar to those recorded in fish kept in hyposmotic environments. Although some of the morpho-physiological mechanisms were operational in juvenile Siberian sturgeon in their adaptation to hyperosmotic media, fish cannot be considered hyperosmotic regulators as they were unable to maintain their plasma osmolality and electrolyte equilibrium in salinities higher than 250 mOsmol kg−1. This suggests that the culture of juvenile Siberian sturgeon in brackish environments is unlikely to be successful. However, our data indicate that in natural environments, juvenile Siberian sturgeon in migratory populations (Ob and Lena Rivers) would be able to migrate successfully into estuarine brackish grounds with a salinity of up to 9%.

All functions in animals rely on daily rhythms, and mealtime can act as a rhythm-marker of nutrients assimilation and use. The effects of meal timing and food composition on carbohydrate use and protein retention of gilthead sea bream were studied. Three groups were fed twice a day (10 am and at 5 pm) for two months with two alternating diets: a commercial diet (Cd) and a high-carbohydrate, low-protein diet (Ed). The Ed/Cd group received the Ed diet in the morning and the Cd diet in the afternoon, and the Cd/Ed group received these diets in the reverse order. The Cd/Cd group only received the commercial diet (control group). After 56 days, two force-feeding experiments (PF1 and PF2) measured for all three groups the fate of a single meal labelled with 15N-protein and 13C-starch through the retention of both isotopes in the main organs and tissue reserves. In PF1 fish were fed at 10 am (morning mealtime), and in PF2 at 5 pm (afternoon mealtime). Fish were sampled at the next two mealtimes (PF1: 7 and 24 h post-feeding, PF2: 17 and 24 h post-feeding). Nutrients recovery differed according to, first, the dietary regime, and second, the last meal received (Cd or Ed). Daily lower protein intake was compensated with higher protein retention combined with more use of carbohydrates for energy. Nevertheless, carbohydrates from the morning meal were used more efficiently. So, the use of carbohydrate for energy production and protein for growth can be improved by adjusting diet composition and mealtime.

Training at sustainable swimming speeds can produce changes in fish skeletal muscle that are important for aquaculture due to their growth-potentiating effects. Such changes may be even more relevant when fish are fed diets containing an increasing proportion of carbohydrates as an energy source. We evaluated the effects of moderate-intensity sustained swimming on the transcriptomic response of red and white muscle in rainbow trout fed a carbohydrate-rich diet using microarray and qPCR. Analysis of the red and white muscle transcriptome in resting or swimming (1.3 body lengths/s) for 30 days revealed significant changes in the expression of a large number of genes (395 and 597, respectively), with a total of 218 differentially expressed genes (DEGs) common for both muscles. A large number of the genes involved in glucose use and energy generation, contraction, development, synthesis and catabolism of proteins were up-regulated in red and white muscle. Additionally, DEGs in both muscles were involved in processes of defense response and apoptosis. Skeletal muscle contraction activates a transcriptional program required for the successful adaptation of both muscles to the changing demands imposed by swimming conditions. Future studies should further clarify the mechanisms involved in the adaptation of both tissues to exercise and assess possible benefits of such conditions for cultured fish. Fil: Magnoni, Leonardo Julián. Universidad de Barcelona. Facultad de Biología; España. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Crespo, Diego. Universidad de Barcelona. Facultad de Biología; España Fil: Ibarz, Antoni. Universidad de Barcelona. Facultad de Biología; España Fil: Blasco, Josefina. Universidad de Barcelona. Facultad de Biología; España Fil: Fernández Borrás, Jaume. Universidad de Barcelona. Facultad de Biología; España Fil: Planas, Josep V.. Universidad de Barcelona. Facultad de Biología; España