Acute Stress in Lesser-Spotted Catshark (Scyliorhinus canicula Linnaeus, 1758) Promotes Amino Acid Catabolism and Osmoregulatory Imbalances.

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.

Cortisol and Dexamethasone Mediate Glucocorticoid Actions in the Lesser Spotted Catshark (Scyliorhinus canicula).

Corticosteroids are hormones produced in vertebrates exerting gluco- and mineralocorticoid actions (GC and MC) mediated by specific receptors (GR and MR, respectively). In elasmobranchs, the major circulating corticosteroid is the 1α-hydroxycorticosterone (1α-OHB). This hormone acts as a MC, but to date its role as a GC has not been established. As there is no 1α-OHB standard available, here we employed a set of in vivo and ex vivo approaches to test GC actions of other corticosteroids in the lesser spotted catshark (Scyliorhinus canicula). Dexamethasone (DEX, a synthetic corticosteroid) slow-release implants decreased plasma 1α-OHB levels after 7 days, and modified carbohydrates metabolism in liver and white muscle (energy stores and metabolic enzymes). In addition, ex vivo culture of liver and white muscle explants confirmed GC actions of corticosteroids not naturally present in sharks (cortisol and DEX) by increasing glucose secretion from these tissues. Dose-response curves induced by cortisol and DEX, altogether with the use of specific GR inhibitor mifepristone, confirmed the involvement of GR mediating glucose secretion. This study highlights the influence of corticosteroids in the glucose balance of S. canicula, though the role of 1α-OHB as a GC hormone in sharks should be further confirmed.

Water temperature affects osmoregulatory responses in gilthead sea bream (Sparus aurata L.).

Sea bream (Sparus aurata Linneaus) was acclimated to three salinity concentrations, viz. 5 (LSW), 38 (SW) and 55psµ (HSW) and three water temperatures regimes (12, 19 and 26 °C) for five weeks. Osmoregulatory capacity parameters (plasma osmolality, sodium, chloride, cortisol, and branchial and renal Na+,K+-ATPase activities) were also assessed. Salinity and temperature affected all of the parameters tested. Our results indicate that environmental temperature modulates capacity in sea bream, independent of environmental salinity, and set points of plasma osmolality and ion concentrations depend on both ambient salinity and temperature. Acclimation to extreme salinity resulted in stress, indicated by elevated basal plasma cortisol levels. Response to salinity was affected by ambient temperature. A comparison between branchial and renal Na+,K+-ATPase activities appears instrumental in explaining salinity and temperature responses. Sea bream regulate branchial enzyme copy numbers (Vmax) in hyperosmotic media (SW and HSW) to deal with ambient temperature effects on activity; combinations of high temperatures and salinity may exceed the adaptive capacity of sea bream. Salinity compromises the branchial enzyme capacity (compared to basal activity at a set salinity) when temperature is elevated and the scope for temperature adaptation becomes smaller at increasing salinity. Renal Na+,K+-ATPase capacity appears fixed and activity appears to be determined by temperature.

Contribution of Non-canonical Cortisol Actions in the Early Modulation of Glucose Metabolism of Gilthead Sea Bream (Sparus aurata).

Teleost fish are exposed to diverse stressors in farming and wildlife conditions during their lifespan. Cortisol is the main glucocorticoid hormone involved in the regulation of their metabolic acclimation under physiological stressful conditions. In this context, increased plasma cortisol is associated with energy substrate mobilization from metabolic tissues, such as liver and skeletal muscle, to rapidly obtain energy and cope with stress. The metabolic actions of cortisol have primarily been attributed to its genomic/classic action mechanism involving the interaction with intracellular receptors, and regulation of stress-responsive genes. However, cortisol can also interact with membrane components to activate rapid signaling pathways. In this work, using the teleost fish gilthead sea bream (Sparus aurata) as a model, we evaluated the effects of membrane-initiated cortisol actions on the early modulation of glucose metabolism. For this purpose, S. aurata juveniles were intraperitoneally administrated with cortisol and with its membrane impermeable analog, cortisol-BSA. After 1 and 6 h of each treatment, plasma cortisol levels were measured, together with glucose, glycogen and lactate in plasma, liver and skeletal muscle. Transcript levels of corticosteroids receptors (gr1, gr2, and mr) and key gluconeogenesis (g6pc and pepck)- and glycolysis (pgam1 and aldo) related genes in the liver were also measured. Cortisol and cortisol-BSA administration increased plasma cortisol levels in S. aurata 1 h after administration. Plasma glucose levels enhanced 6 h after each treatment. Hepatic glycogen content decreased in the liver at 1 h of both cortisol and cortisol-BSA administration, while increased at 6 h due to cortisol but not in response to cortisol-BSA. Expression of gr1, g6pc, pgam1, and aldo were preferentially increased by cortisol-BSA in the liver. Taking all these results in consideration, we suggest that non-canonical cortisol mechanisms contribute to the regulation of the early glucose metabolism responses to stress in S. aurata.

Plasma 1α-Hydroxycorticosterone as Biomarker for Acute Stress in Catsharks (Scyliorhinus canicula).

Glucocorticoids are pleiotropic steroid hormones mediating redistribution of energy. They induce breakdown of glycogen stores and consequent plasma hyperglycaemia after stressful situations. Glucocorticoid actions in most vertebrate species are exerted by cortisol and corticosterone. However, 1α-hydroxycorticosterone is the dominant corticosteroid hormone in elasmobranchs, though its effects as a glucocorticoid are unknown. Here we demonstrate, by using ultra-performance liquid chromatography coupled to tandem mass spectrometry for the quantification of 1α-hydroxycorticosterone in plasma of the elasmobranch Scyliorhinus canicula, the response of this hormone to an acute-stress situation and for the first time its glucocorticoid action in elasmobranchs. After an acute air-exposure challenge, S. canicula increased plasma levels of 1α-hydroxycorticosterone altogether with enhanced glycolysis and gluconeogenesis pathways to fuel energy demanding tissues, such as white muscle, during the first hours after the stress situation. We foresee our study as a starting point to evaluate stress responses in elasmobranchs, as well as for future applications in the management of these key ecosystem species.

Metabolic and Stress Responses in Senegalese Soles (Solea senegalensis Kaup) Fed Tryptophan Supplements: Effects of Concentration and Feeding Period.

: The objective of this study was to assess the impact of different dietary Trp concentrations on the stress and metabolism response of juvenile Senegalese soles (Solea senegalensis). Fish (38.1 ± 1.9 g) were fed different Trp-enriched feeds (0%, 1% and 2% Trp added) for two and eight days, and later exposed to air stress for three min. Samples were taken pre- and 1 h post-stress (condition). Plasma cortisol, lactate, glucose and proteins were significantly affected by the sampling time, showing higher values at 1 h post-stress. Trp concentration in food also had significant effects on lactate and glucose levels. However, the feeding period did not affect these parameters. Post-stress values were higher than in the pre-stress condition for every plasma parameter, except for lactate in two days and 1% Trp treatment. Nevertheless, cortisol, glucose and lactate did not vary significantly between pre- and post-stress samplings in fish fed the 1% Trp-enriched diet for two days. The lack of variability in cortisol response was also due to the high pre-stress value, significantly superior to pre-stress control. The exposure time to Trp feeding did not significantly affect any enzyme activity; however, Trp added and condition influenced protein-related enzyme activities. In spite of decreasing stress markers, Trp-enriched diets altered the protein metabolism.

Dietary Tryptophan Induces Opposite Health-Related Responses in the Senegalese Sole (Solea senegalensis) Reared at Low or High Stocking Densities With Implications in Disease Resistance.

High rearing densities are typical conditions of both inland and onshore intensive aquaculture units. Despite obvious drawbacks, this strategy is nonetheless used to increase production profits. Such conditions inflict stress on fish, reducing their ability to cope with disease, bringing producers to adopt therapeutic strategies. In an attempt to overcome deleterious effects of chronic stress, Senegalese sole, Solea senegalensis, held at low (LD) or high density (HD) were fed tryptophan-supplemented diets with final tryptophan content at two (TRP2) or four times (TRP4) the requirement level, as well as a control and non-supplemented diet (CTRL) for 38 days. Fish were sampled at the end of the feeding trial for evaluation of their immune status, and mortalities were recorded following intra-peritoneal infection with Photobacterium damselae subsp. piscicida. Blood was collected for analysis of the hematological profile and innate immune parameters in plasma. Pituitary and hypothalamus were sampled for the assessment of neuro-endocrine-related gene expression. During the feeding trial, fish fed TRP4 and held at LD conditions presented higher mortalities, whereas fish kept at HD seemed to benefit from this dietary treatment, as disease resistance increased over that of CTRL-fed fish. In accordance, cortisol level tended to be higher in fish fed both supplemented diets at LD compared to fish fed CTRL, but was lower in fish fed TRP4 than in those fed TRP2 under HD condition. Together with lower mRNA levels of proopiomelanocortin observed with both supplementation levels, these results suggest that higher levels of tryptophan might counteract stress-induced cortisol production, thereby rendering fish better prepared to cope with disease. Data regarding sole immune status showed no clear effects of tryptophan on leucocyte numbers, but TRP4-fed fish displayed inhibited alternative complement activity (ACH50) when held at LD, as opposed to their HD counterparts whose ACH50 was higher than that of CTRL-fed fish. In conclusion, while dietary tryptophan supplementation might have harmful effects in control fish, it might prove to be a promising strategy to overcome chronic stress-induced disease susceptibility in farmed Senegalese sole.

Transport and Recovery of Gilthead Sea Bream (Sparus aurata L.) Sedated With Clove Oil and MS222: Effects on Oxidative Stress Status.

The use of anesthesia is a common practice in aquaculture to sedate fish and mitigate handling stress. Although the employ of anesthesia is considered beneficial for fish, as it reduces stress and improves welfare, at the same time it may induce hazardous side-effects. The aim of the present study was to investigate the effects of clove oil (CO) and tricaine methanesulfonate (MS222), two of the most used anesthetics, on several oxidative stress related parameters in gilthead sea bream (Sparus aurata), as these types of effects of anesthetics have been seldom investigated. To assess these effects, S. aurata juveniles were placed in a setup of mobile water tanks and were transported during 6 h with either 2.5 mg/L CO or 5 mg/L MS222. After transport, half of the fish were sampled, whereas the remaining fish were transferred to tanks without anesthetics where they were allowed to recover for 18 h before sampling. Changes in the expression levels of several target genes related with the antioxidant response and cell-tissue repair were evaluated in the gills, liver and brain. Those transcripts included glutathione peroxidase 1 (gpx1), catalase (cat), glutathione S-transferase 3 (gst3), glutathione reductase (gr), superoxide dismutase [Zn] (sod2), heat shock protein-70 (hsp70), and metallothionein (mt). Antioxidant enzymatic activities glutathione S-transferase, GST; catalase, CAT; and glutathione reductase, GR, levels of non-enzymatic antioxidants (non-protein thiols - NPT), and pro-oxidative damage, assessed as lipid peroxidation (LPO), were determined in gills, liver and brain. Acetylcholinesterase activity (AChE) was determined in plasma, gills, brain, muscle and heart as an indicator of neuro-muscular alterations. In plasma, the total antioxidant capacity (TAC) and total oxidative status (TOS) were also measured. Results showed that the use of both anesthetic agents, CO and MS222, interferes with fish antioxidant status. All tested biological matrices displayed alterations in antioxidant endpoints, confirming that these substances, although minimizing the effects of transport stress, may have long term effects on fish defenses. This result is of high relevance to aquaculture considering that the oxidative stress, may increase the susceptibility to different environmental or biotic stress and different types of pathologies.

Impact of Air Exposure on Vasotocinergic and Isotocinergic Systems in Gilthead Sea Bream (Sparus aurata): New Insights on Fish Stress Response.

The hypothalamus-pituitary-interrenal (HPI) and hypothalamus-sympathetic-chromaffin cell (HSC) axes are involved in the regulation of the stress response in teleost. In this regard, the activation of a complex network of endocrine players is needed, including corticotrophin-releasing hormone (Crh), Crh binding protein (Crhbp), proopiomelanocortin (Pomc), thyrotropin-releasing hormone (Trh), arginine vasotocin (Avt), and isotocin (It) to finally produce pleiotropic functions. We aimed to investigate, using the gilthead sea bream (Sparus aurata) as a biological model, the transcriptomic response of different endocrine factors (crh, crhbp, pomcs, trh), neuropeptides (avt and it), and their specific receptors (avtrv1a, avtrv2, and itr) in four important target tissues (hypothalamus, pituitary, kidney and liver), after an acute stress situation. We also investigated several stress hormones (catecholamines and cortisol). The stress condition was induced by air exposure for 3 min, and hormonal, metabolic and transcriptomic parameters were analyzed in a time course response (15 and 30 min, and 1, 2, 4, and 8 h post-stress) in a total of 64 fish (n = 8 fish per experimental group; p = 0.05; statistical power = 95%). Our results showed that plasma noradrenaline, adrenaline and cortisol values increased few minutes after stress exposure. At hypothalamic and hypophyseal levels, acute stress affected mRNA expression of all measured precursors and hormonal factors, as well as their receptors (avtrs and itr), showing the activation, at central level, of HPI, HSC, and Avt/It axes in the acute stress response. In addition, stress response also affected mRNA levels of avtrs and itr in the head kidney, as well as the steroidogenic acute regulatory protein (star) and tyrosine hydroxylase (th) expression, suggesting their participation in the HPI and HSC axes activation. Moreover, the pattern of changes in hepatic avtrs and itr gene expression also highlights an important role of vasotocinergic and isotocinergic pathways in liver metabolic organization after acute stress events. Our results demonstrate, both at transcriptional and circulating levels of several hormones, the existence of a complex activation of different endocrine pathways in S. aurata related to the stress pathways, where vasotocinergic and isotocinergic systems can also be considered key players of the acute stress response orchestration.

Myrcia sylvatica essential oil mitigates molecular, biochemical and physiological alterations in Rhamdia quelen under different stress events associated to transport.

The effects of pre-transport handling and addition of essential oil of Myrcia sylvatica (EOMS) during transport on stress pathways activation in Rhamdia quelen were investigated. Fish (n=400, 25.2±2.9g) were captured in production ponds and transferred to 100-L tank (density 100g L-1). After 24h, 10 fish were sampled (before transport group). The remaining fish were placed in plastic bags (n=30 or 32 fish per bag, density 150g L-1) containing 5L of water (control), ethanol (315µLL-1, vehicle) or EOMS (25 or 35µLL-1), in triplicate, transported for 6h and sampled (n=10 animals per group). Indicators of stress and metabolism, as well as mRNA expression of brain hormones were evaluated. Previously, full-length cDNAs, encoding specific corticotropin-releasing hormone (crh) and proopiomelanocortins (pomca and pomcb), were cloned from whole brain of R. quelen. Crh expression increased after 24h of capture and handling, whereas cortisol and glucose plasmatics enhanced their values in the control group. Transport with EOMS reduced plasma cortisol and lactate levels, while ethanol and EOMS groups increased Na+/K+-ATPase gill activity compared to control. Gene expression of crh, pomcb, prolactin and somatolactin mRNAs were lower after transport with EOMS compared to control. EOMS was able to mitigate the stress pathways activation caused by transport, maintaining a balance in body homeostasis. Thus, EOMS is recommended as sedative in procedures as transport and the pre-transport handling requires greater attention and use of tranquilizers.

Carbohydrates digestion and metabolism in the spiny lobster (Panulirus argus): biochemical indication for limited carbohydrate utilization.

As other spiny lobsters, Panulirus argus is supposed to use preferentially proteins and lipids in energy metabolism, while carbohydrates are well digested but poorly utilized. The aim of this study was to evaluate the effect of dietary carbohydrate level on digestion and metabolism in the spiny lobster P. argus. We used complementary methodologies such as post-feeding flux of nutrients and metabolites, as well as measurements of α-amylase expression and activity in the digestive tract. Lobsters readily digested and absorbed carbohydrates with a time-course that is dependent on their content in diet. Lobster showed higher levels of free glucose and stored glycogen in different tissues as the inclusion of wheat flour increased. Modifications in intermediary metabolism revealed a decrease in amino acids catabolism coupled with a higher use of free glucose as carbohydrates rise up to 20%. However, this effect seems to be limited by the metabolic capacity of lobsters to use more than 20% of carbohydrates in diets. Lobsters were not able to tightly regulate α-amylase expression according to dietary carbohydrate level but exhibited a marked difference in secretion of this enzyme into the gut. Results are discussed to highlight the limitations to increasing carbohydrate utilization by lobsters. Further growout trials are needed to link the presented metabolic profiles with phenotypic outcomes.

Energy metabolism of hyperthyroid gilthead sea bream Sparus aurata L.

Thyroid hormones, in particular 3,5,3'-triiodothyronine or T3, are involved in multiple physiological processes in mammals such as protein, fat and carbohydrate metabolism. However, the metabolic actions of T3 in fish are still not fully elucidated. We therefore tested the effects of T3 on Sparus aurata energy metabolism and osmoregulatory system, a hyperthyroid-induced model that was chosen. Fish were implanted with coconut oil depots (containing 0, 2.5, 5.0 and 10.0µg T3/g body weight) and sampled at day 3 and 6 post-implantation. Plasma levels of free T3 as well as glucose, lactate and triglyceride values increased with increasing doses of T3 at days 3 and 6 post-implantation. Changes in plasma and organ metabolite levels (glucose, glycogen, triglycerides, lactate and total α amino acid) and enzyme activities related to carbohydrate, lactate, amino acid and lipid pathways were detected in organs involved in metabolism (liver) and osmoregulation (gills and kidney). Our data implicate that the liver uses amino acids as an energy source in response to the T3 treatment, increasing protein catabolism and gluconeogenic pathways. The gills, the most important extruder of ammonia, are fuelled not only by amino acids, but also by lactate. The kidney differs significantly in its substrate preference from the gills, as it obtained metabolic energy from lactate but also from lipid oxidation processes. We conclude that in S. aurata lipid catabolism and protein turnover are increased as a consequence of experimentally induced hyperthyroidism, with secondary osmoregulatory effects.

Cortisol modulates vasotocinergic and isotocinergic pathways in the gilthead sea bream.

In the present study, we assessed the responses of the vasotocinergic and isotocinergic systems to chronic stress induced by cortisol administration in the gilthead sea bream (Sparus aurata). Pituitary and plasma arginine vasotocin (AVT) and isotocin (IT) levels, as well as hypothalamic pro-vasotocin (pro-VT) and pro-isotocin (pro-IT) mRNA expression levels, were analysed. In addition, the mRNA levels of three receptors, AVTR type V1a2, AVTR type V2 and ITR, were analysed in several target organs associated with the following physiological processes: (i) integration and control (hypothalamus), (ii) metabolism and its control (liver and hypothalamus), (iii) osmoregulation (gills) and (iv) stress response (head kidney). Specimens were injected intraperitoneally with slow-release implants (5 µL g(-1) body mass) containing coconut oil alone (control group) or with cortisol (50 µg g(-1) body mass; cortisol group). Both AVT and IT synthesis and release were correlated with plasma cortisol values, suggesting a potential interaction between both hormonal systems and cortisol administration. Our results suggest that the activation of hepatic metabolism as well as the hypothalamic control of metabolic processes provide the energy necessary to overcome stress, which could be partly mediated by AVTRs and ITR. Upregulation of branchial AVT and IT receptor expression following cortisol treatment suggests an involvement of the vasotocinergic and isotocinergic systems in the regulation of ion channels/transporters during stressful situations. Finally, changes in AVT and IT receptor mRNA expression in the head kidney suggest these nonapeptides participate in feedback mechanisms that regulate the synthesis/release of cortisol. Our results indicate a relationship between cortisol and both the vasotocinergic and isotocinergic systems during simulated chronic stress in S. aurata.

Different stressors induce differential responses of the CRH-stress system in the gilthead sea bream (Sparus aurata).

The hypothalamus-pituitary-interrenal (HPI) axis, involved in the regulation of the neuroendocrine stress responses, presents important players such as corticotropin-releasing hormone (CRH, generally considered as the initiator of this pathway) and CRH-binding protein (CRH-BP, considered as an antagonist of CRH function). CRH and CRH-BP full-length cDNA sequences were obtained from Sparus aurata by screening a brain cDNA library, and their phylogenetic analysis as well as their roles during acute and chronic stress responses were assessed. mRNA expression levels and plasma cortisol concentrations were measured by RT qPCR and ELISA, respectively, in S. aurata juveniles submitted to: i) different environmental salinities in a short-time course response; and ii) food deprivation during 21 days. In addition, osmoregulatory and metabolic parameters in plasma corroborated a clear reorganization depending on the stress source/period. Salinity transfer induced stress as indicated by enhanced plasma cortisol levels, as well as by up-regulated CRH and down-regulated CRH-BP expression values. On the other hand, food deprivation did not affect both expression levels, although plasma cortisol concentrations were enhanced. These results suggest that different stressors are handled through different stress pathways in S. aurata.

Temperature enhanced effects of chlorine exposure on the health status of the sentinel organism Mytilus galloprovincialis.

It now is widely recognised that the global temperature is rising, a phenomenon which could alter the effects of pollution on wildlife. In order to assess the role of temperature and exposure to chlorine due to cooling water discharges, a battery of metabolic, oxidative stress and histological parameters were evaluated in Mytilus galloprovincialis after 15 and 30 days at 15 °C and at two increased temperatures (+5 and +10 °C). Diverse gill pathologies such as haemolymphatic sinus dilatation, an increased number of mucocytes and granulocytes as well as a lower number of cilia were observed after 30 days exposure at higher temperatures. Protein, amino acid, triglyceride and fatty acid levels decreased when the temperature increased, as a consequence of higher energetic demand. Similarly, acetylcholinesterase, catalase and glutathione S-transferase activities showed an inhibition at higher temperatures, although gill lipid peroxidation levels remained unaffected. Our results suggest that increased temperatures induce deterioration in the health status of the mussels and in their defensive capacity against a polluted environment.

Lobster (Panulirus argus) hepatopancreatic trypsin isoforms and their digestion efficiency.

It is well known that crustaceans exhibit several isoforms of trypsin in their digestive system. Although the number of known crustacean trypsin isoforms continues increasing, especially those derived from cDNA sequences, the role of particular isoenzymes in digestion remains unknown. Among invertebrates, significant advances in the understanding of the role of multiple trypsins have been made only in insects. Since it has been demonstrated that trypsin isoenzyme patterns (phenotypes) in lobster differ in digestion efficiency, we used this crustacean as a model for assessing the biochemical basis of such differences. We demonstrated that the trypsin isoform known to be present in all individuals of Panulirus argus has a high catalytic efficiency (k(cat)/K(m) ) and is the most reactive toward native proteinaceous substrates, whereas one of the isoforms present in less efficient individuals has a lower k(cat) and a lower k(cat)/K(m), and it is less competent at digesting native proteins. A fundamental question in biology is how genetic differences produce different physiological performances. This work is the first to demonstrate that trypsin phenotypic variation in crustacean protein digestion relies on the biochemical properties of the different isoforms. Results are relevant for understanding trypsin polymorphism and protein digestion in lobster.

Food deprivation induces chronic stress and affects thyroid hormone metabolism in Senegalese sole (Solea senegalensis) post-larvae.

In vertebrates, stress and thyroid systems interact closely, most likely because of the involvement of both systems in energy metabolism. However, studies on these interactions, especially during larval development, are scarce. Recently, cDNAs coding for corticotropin-releasing hormone (CRH) and CRH-binding protein (CRH-BP), two key players in the regulation of the neuroendocrine stress response, were characterized for the flatfish Senegalese sole (Solea senegalensis). To investigate the involvement of stress and thyroid systems in this species, the effects of food deprivation during early development of S. senegalensis were assessed. Growth was arrested in food-deprived post-larvae, which was also reflected by decreased carbon and nitrogen contents, indicating increased catabolism. Food deprivation induces chronic stress, as illustrated by enhanced whole-body cortisol levels, as well as an up regulation of crh and a decrease of crh-bp expression levels. Furthermore, whole-body total T3 concentrations of food-deprived post-larvae were reduced, although tshß subunit expression levels remained unaffected. Our results show that food deprivation is a chronic stressor that induces energy-releasing catabolic processes that compensate for the reduced energy intake, and inhibits anabolic processes via the peripheral thyroid system.

Dietary protein quality differentially regulates trypsin enzymes at the secretion and transcription level in Panulirus argus by distinct signaling pathways.

The effects of pelleted diets with different protein composition (fish, squid or soybean meals as main protein sources) on trypsin secretion and expression were studied in the lobster Panulirus argus. Trypsin secretion was shown to be maximal 4 h after ingestion. At this time, fish- and squid-based diets induced trypsin secretion, as well as up-regulation of the major trypsin isoform at the transcription level. While fish- and squid-based diets elicited a prandial response, soybean-based diet failed to stimulate the digestive gland to secrete trypsin into the gastric fluid or induce trypsin expression above the levels observed in fasting lobsters. In vitro assays showed that intact proteins rather than protein hydrolysates stimulate trypsin secretion in the lobster. However, the signal for trypsin transcription appears to be different to that for secretion and is probably mediated by the appearance of free amino acids in the digestive gland, suggesting a stepwise regulation of trypsin enzymes during digestion. We conclude that trypsin enzymes in P. argus are regulated at the transcription and secretion level by the quality of dietary proteins through two distinct signaling pathways. Our results indicate that protein digestion efficiency in spiny lobsters can be improved by selecting appropriated protein sources. However, other factors like the poor solubility of dietary proteins in dry diets could hamper further enhancement of digestion efficiency.

Different environmental temperatures affect amino acid metabolism in the eurytherm teleost Senegalese sole (Solea senegalensis Kaup, 1858) as indicated by changes in plasma metabolites.

Senegalese sole (Solea senegalensis) is a eurytherm teleost that under natural conditions can be exposed to annual water temperature fluctuations between 12 and 26°C. This study assessed the effects of temperature on sole metabolic status, in particular in what concerns plasma free amino acid changes during thermal acclimation. Senegalese sole maintained at 18°C were acclimated to either cold (12°C) or warm (26°C) environmental temperatures for 21 days. Fish maintained at 18°C served as control. Plasma concentrations of cortisol, glucose, lactate, triglycerides, proteins, and free amino acids were assessed. Cold acclimation influenced interrenal responses of sole by increasing cortisol release. Moreover, plasma glucose and lactate concentrations increased linearly with temperature, presumably reflecting a higher metabolic activity of sole acclimated to 26°C. Acclimation temperature affected more drastically plasma concentrations of dispensable than that of indispensable amino acids, and different acclimation temperatures induced different responses. Asparagine, glutamine and ornithine seem to be of particular importance for ammonia detoxification mechanisms, synthesis of triglycerides that may be used during homeoviscous adaptation and, to a lesser extent, as energetic substrates in specimens acclimated to 12°C. When sole is acclimated to 26°C taurine, glutamate, GABA and glycine increased, which may suggest important roles as antioxidant defences, in osmoregulatory processes and/or for energetic purposes at this thermal regimen. In conclusion, acclimation to different environmental temperatures induces several metabolic changes in Senegalese sole, suggesting that amino acids may be important for thermal acclimation.

Subfunctionalization of POMC paralogues in Senegalese sole (Solea senegalensis).

The precursor protein proopiomelanocortin (POMC) gives rise to a variety of biologically active peptides through cell-specific posttranslational processing. Two transcripts of pomc were found in the flatfish Solea senegalensis (ssePOMC-A and ssePOMC-B), that most likely represent subfunctionalized paralogues: ssePOMC-A lacks the N-terminal cleavage site for ß-MSH, whereas ssePOMC-B cannot yield ACTH and completely lacks the opioid consensus sequence in the ß-END region. An analysis of nucleotide substitution rates shows that the POMC-derived peptides possess well-conserved regions under purifying selection, except the ß-END derived from POMC-B, which has undergone positive selection. The calculated K(s) values for ssePOMC-A versus ssePOMC-B and zebrafish POMCαversus zebrafish POMCß are 0.40 and 0.72, respectively, indicating that the zebrafish POMC paralogues started to evolve almost twice as early in evolution, and that the Solea POMC paralogues arose independently from the whole genome duplication event that gave rise to the zebrafish paralogues. This makes ssePOMC-B the first identified POMCα orthologue that lacks the opioid consensus. Furthermore, pomc-a expression is down-regulated in chronic stressed S. senegalensis juveniles, whereas pomc-b expression levels remain unaffected, indicating different physiological roles for both POMC paralogues. The distribution of functional POMC-derived peptide hormones over two pomc genes in S. senegalensis suggests subfunctionalization of the paralogues, a relevant notion when studying POMC function in endocrine responses.