In vivo regulation of GLUT2 mRNA in sea bass (Dicentrarchus labrax) in response to acute and chronic hypoxia

The expression and regulation of sodium-independent glucose transporter (GLUT)-2, in relation to hypoxia has not yet been explored in fish or other vertebrates. In this study, the complete open-reading frame for sea bass GLUT2 was isolated and deposited in the GenBank. The predicted 12 transmembrane domains of the protein (508 amino acids) are presented. A phylogenetic tree was constructed on GLUT2 sequences of sea bass and those of other teleost, amphibian, avian, and mammalian species. We also analyzed acute and chronic hypoxia-induced changes in the expression of hepatic GLUT2 mRNA, using one-tube, two-temperature, real-time RT-PCR with which gene expression can be absolutely quantified by the standard curve method. The number of GLUT2 mRNA copies was significantly increased in response to both acute (1.9 mg/L, dissolved oxygen for 4 h) and chronic (4.3 mg/L, DO for 15 days) hypoxia conditions. The hypoxia-related changes in GLUT2 mRNA copy number support the view that GLUT2 is involved in the adaptation response to hypoxia in sea bass, a marine hypoxia-sensitive species. We realize that the GLUT2 mRNA levels in our study do not measure the physiological effects produced by the protein. Thus, we can only speculate that, under hypoxic conditions, GLUT2 probably functions to allow the glucose produced from liver glycogen to leave the hepatocytes.     

Effects of dopaminergic system activation on feeding behavior and growth performance of the sea bass (Dicentrarchus labrax): A self-feeding approach

Dopamine is synthesized from l-dopa and subsequently processed into norepinephrine and epinephrine. Any excess neurotransmitter can be taken up again by the neurons to be broken down enzymatically into DOPAC. The effect of dopamine on mammalian food intake is controversial. Mice unable to synthesize central dopamine die of starvation. However, studies have also shown that central injection of dopamine inhibits food intake. The effect of dopaminergic system in the fish feeding behavior has been scarcely explored. We report that the inclusion of l-dopa in the diets results in the activation of sea bass central dopaminergic system but also in the significant increase of the hypothalamic serotonin levels. Dietary l-dopa induces a decrease of food intake and feed conversion efficiency that drives a decline of all growth parameters tested. No behavioral effects were observed after l-dopa treatment. l-dopa treatment stimulated central expression of NPY and CRF. It suggests that CRF might mediate l-dopa effects on food intake but also that CRF neurons lie downstream of NPY neurons in the hierarchical forebrain system, thus controlling energy balance. Unexpectedly, dietary administration of haloperidol, a D₂-receptor antagonist, cannot block dopamine effects but also induces a decline of the food intake. This decrease seems to be a side effect of haloperidol treatment since fish exhibited a decreased locomotor activity. We conclude that oral l-dopa inhibits sea bass food intake and growth. Mechanism could also involve an increase of hypothalamic serotoninergic tone.     

Effects of water salinity on melatonin levels in plasma and peripheral tissues and on melatonin binding sites in European sea bass (Dicentrarchus labrax)

Sea bass is an euryhaline fish that lives in a wide range of salinities and migrates seasonally from lagoons to the open sea. However, to date, the influence of water salinity on sea bass melatonin levels has not been reported. Here, we evaluated the differences in plasma and tissue melatonin contents and melatonin binding sites in sea bass under four different salinity levels: seawater (36‰), isotonic water (15‰), brackish water (4‰) and freshwater (0‰). The melatonin content was evaluated in plasma, whole brain, gills, intestine and kidney, while melatonin binding sites were analyzed in different brain regions and in the neural retina. Plasma melatonin levels at mid-dark varied, the lowest value occurring in seawater (102 pg/mL), and the highest in freshwater (151 pg/mL). In gills and intestine, however, the highest melatonin values were found in the seawater group (209 and 627 pg/g tissue, respectively). Melatonin binding sites in the brain also varied with salinity, with the highest density observed at the lower salinities in the optic tectum, cerebellum and hypothalamus (30.3, 13.0, and 8.0 fmol/mg protein, respectively). Melatonin binding sites in the retina showed a similar pattern, with the highest values being observed in freshwater. Taken together, these results reveal that salinity influences melatonin production and modifies the density of binding sites, which suggests that this hormone could play a role in timing seasonal events in sea bass, including those linked to fish migration between waters of different salinities for reproduction and spawning.     

Effect of conjugated linoleic acid on dietary lipids utilization, liver morphology and selected immune parameters in sea bass juveniles (Dicentrarchus labrax)

Increased energy content in fish feeds has led to an enhanced fat deposition, particularly in European sea bass, concerning fish farmers. Inclusion of conjugated linoleic acid (CLA) could reduce fat deposition as in other vertebrates. To determine if dietary CLA affects fat deposition, lipid metabolism, lipid composition and morphology of different tissues, growth and selected immune parameters, European sea bass juveniles were fed 4 graded levels of CLA (0, 0.5, 1 and 2%). Growth and feed conversion were not affected by CLA, whereas feed intake was reduced (P < 0.05) by feeding 2% CLA. In these fish perivisceral fat was also reduced (P < 0.05), particularly reducing (P < 0.05) monounsaturated fatty acids. CLA has not affected tissue proximal composition, but reduced (P < 0.05) saturated and monounsaturated fatty acids and increased (P < 0.05) the n−3 and n−3 highly unsaturated fatty acids in muscle and increase (P < 0.05) CLA content in muscle, liver and perivisceral fat. A progressive reduction in lipid vacuolization of hepatocytes cytoplasm and regular-shaped morphology was found in fish fed increased CLA levels, together with a progressive increase in malic enzyme activity (only significant in fish fed 1% CLA). Finally, inclusion of CLA up to 1% increased (P < 0.05) plasma lysozyme activity and was positively correlated with alternative complement pathway.     

Postprandial gastrointestinal blood flow, oxygen consumption and heart rate in rainbow trout (Oncorhynchus mykiss)

The present study is the first to simultaneously and continuously measure oxygen consumption (MO₂) and gastrointestinal blood flow (q_gi) in fish. In addition, while it is the first to compare the effects of three isoenergetic diets on q_gi in fish, no significant differences among diets were found for postprandial MO₂, q_gi or heart rate (f_H) in rainbow trout, Oncorhynchus mykiss. Postprandial q_gi, f_H and MO₂ were significantly elevated above baseline levels by 4 h. Postprandial q_gi peaked at 136% above baseline after 11 h, f_H peaked at 110% above baseline after 14 h and MO₂ peaked at 96% above baseline after 27 h. Moreover, postprandial MO₂ remained significantly elevated above baseline longer than q_gi (for 41 h and 30 h, respectively), perhaps because most of the increase in MO₂ associated with feeding is due to protein handling, a process that continues following the absorption of nutrients which is thought to be the primary reason for the elevation of q_gi. In addition to the positive relationships found between postprandial MO₂ and q_gi and between postprandial MO₂ and f_H, we discovered a novel relationship between postprandial q_gi and f_H.     

Effects of acute hypoxia and CO2 inhalation on systemic and peripheral oxygen uptake and circulatory responses during moderate exercise

The effect of acute hypoxia and CO2 inhalation on leg blood flow (LBF), on leg vascular resistance (LVR) and on oxygen supply to and oxygen consumption in the exercising leg was studied in nine healthy male subjects during moderate one-leg exercise. Each subject exercised for 20 min on a cycle ergometer in four different conditions: normoxia, normoxia + 2% CO2, hypoxia corresponding to an altitude of 4000 m above sea level, and hypoxia + 1.2% CO2. Gas exchange, heart rate (HR), arterial blood pressure, and LBF were measured, and arterial and venous blood samples were analysed for PCO2, PO2, oxygen saturation, haematocrit and haemoglobin concentration. Systemic oxygen consumption was 1.83 l.min-1 (1.48-2.59) and was not affected by hypoxia or CO2 inhalation in hypoxia. HR was unaffected by CO2, but increased from 136 beat.min-1 (111-141) in normoxia to 155 (139-169) in hypoxia. LBF was 6.5 l.min-1 (5.4-7.6) in normoxia and increased significantly in hypoxia to 8.4 (5.9-10.1). LVR decreased significantly from 2.23 kPa.l-1.min (1.89-2.99) in normoxia to 1.89 (1.53-2.52) in hypoxia. The increase in LBF from normoxia to hypoxia correlated significantly with the decrease in LVR. When CO2 was added in hypoxia a significant correlation was also found between the decrease in LBF and the increase in LVR. In normoxia, the addition of CO2 caused a significant increase in mean blood pressure. Oxygen consumption in the exercising leg (leg VO2) in normoxia was 0.97 l.min-1 (0.72-1.10), and was unaffected by hypoxia and CO2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen uptake,heart rate and activities of locomotor muscles during a critical swimming speed protocol in the gilthead sea bream Sparus aurata

Oxygen uptake, heart rate and contraction frequencies of slow oxidative (SO) and fast glycolytic (FG) muscle were measured simultaneously in gilthead seabream Sparus aurata submitted to stepwise increases in current speed in a swimming respirometer. Variation in oxygen uptake was closely related to variation in heart rate, over initial steps these rose in concert with an increase in contraction frequency of SO muscle. There was an asymptote in oxygen uptake and heart rate at high speeds that reflected a transition from exclusive use of aerobic SO muscle to a combination of SO and anaerobic FG muscle, and which preceded fatigue.     

Gastrointestinal blood flow in the red Irish lord, Hemilepidotus hemilepidotus: long-term effects of feeding and adrenergic control

Cardiac output, blood flow to the coeliac and mesenteric arteries, dorsal aortic blood pressure and heart rate were recorded simultaneously at rest and postprandial for 6 days in a teleost, the red Irish lord (Hemilepidotus hemilepidotus). We anticipated that gastrointestinal blood flow would increase postprandially, supported by an increase in cardiac output. However, we had no predictions for either the exact time-course of this response, or for the regional distribution of blood flow between to the two major arteries comprising the splanchnic circulation. In resting, unfed animals, blood flow to the coeliac artery and mesenteric artery was 4.1 ± 0.6 ml min⁻¹ kg⁻¹ and 4.9 ± 1.3 ml min⁻¹ kg⁻¹, respectively (mean ± SEM, n=7), which together represented 34% of cardiac output. Feeding increased blood flow to the coeliac and mesenteric arteries in a time-dependent manner. The increase in coeliac artery blood flow preceded that in the mesenteric artery, a finding that is consistent with the coeliac artery supplying blood to the liver and stomach, while the mesenteric artery supplies blood to the stomach and intestine. Coeliac blood flow had increased by 84 ± 18% after 1 day and had a peak increase of 112 ± 40% at day 4 postprandial. Mesenteric blood flow was not significantly elevated at day 1, but had increased by 94 ± 19% at day 4 postprandial. Cardiac output also increased progressively, increasing by a maximum of 90 ± 30% at day 4. Because the increase in cardiac output was adequate to meet the postprandial increase in gut blood flow, the postprandial decreases in vascular resistance for the coeliac and mesenteric circulations mirrored the increases in blood flow. Intra-arterial injections of adrenaline and noradrenaline into resting fish more than doubled coeliac and mesenteric vascular resistances, and blood flow decreased proportionately. This adrenergic vasoconstriction was totally abolished by pretreatment with the α-adrenoceptor antagonist phentolamine, which in itself approximately halved coeliac and mesenteric vascular resistances. These observations indicate a significant α-adrenergic tone in the gastrointestinal circulation of the red Irish lord, the loss of which could not entirely account for the postprandial increase in gastrointestinal blood flow. Other control mechanisms are suggested. Accepted: 17 November 1999     

Effects of starvation on valine and alanine transport across the intestinal mucosal border in sea bass,Dicentrarchus labrax

Summary kinetics of intestinal transport of l-alanine and l-valine (substrates of the A-system and the L-system, respectively, in mammals) across the brush-border membrane in sea bass, Dicentrarchus labrax, were studied on intact mucosa using a short-term uptake technique. When fish were starved for 4–8 weeks, total influx (mucosa-to-cell) of valine fell owing to disappearance or modification of the diffusion component. The maximum influx rate of saturable component increased but its affinity (reflected by the Michaelis constant) decreased. Alanine transport by Na⁺-dependent and diffusion pathways was unchanged after starvation Fasting also induced an almost 20% decrease in the length of intestinal microvilli.Abbreviations K _d diffusional constant - K _m Michaelis constant - V _max maximum influx rate     

Nutritional condition, starvation status and growth of early juvenile Japanese sea bass (Lateolabrax japonicus) related to prey distribution and feeding in the nursery ground

The nutritional condition and protein growth rates of Japanese temperate bass larvae and juveniles were studied in relation to prey distribution and feeding habits in the nursery grounds in Chikugo estuary, Ariake Sea, Japan. Samples were collected from a wide spatial area covering the nursery grounds of the fish in March and April 2003. Food habits of the fish were analyzed by examining the gut contents. Fish condition was evaluated by using RNA/DNA ratio and other nucleic acid-based indices and protein growth rates. The nucleic acid contents in individually frozen larvae and juveniles were quantified by fluorometric method. Two distinguished feeding patterns, determined by the distribution of prey copepods, were identified along the nursery ground. The first pattern showed the dependency of the fish on the calanoid copepod Sinocalanus sinensis, which was the single dominant prey in low saline upper river areas and the second pattern involved a multi-species dietary habit mainly dominated by Acartia omorii, Oithona davisae and Paracalanus parvus. Values of RNA, DNA, total protein, growth rates and for all the nucleic acid-based indices were higher in upstream areas than in the downstream areas. The proportion of the starving fish was higher in the downstream areas than in the upstream areas. Condition of juvenile sea bass was not equal throughout the nursery grounds; fish in the upper river were in better condition than those in the lower estuary. We speculated that utilization of S. sinensis, which appears a suitable prey item and provide a better foraging environment in the upstream nursery ground, is one of the key factors for early survival and growth of Japanese temperate bass larvae and juveniles in the Chikugo estuary.     

流速对光棘球海胆行为和生长的影响

海胆在海藻床生态系统结构和功能调控中发挥重要作用。深入理解流速对海胆摄食和生长的影响具有重要的生态学研究价值。研究了长时间(49 d)不同流速(2 cm/s, 10 cm/s和20 cm/s)对光棘球海胆幼胆(Mesocentrotus nudus,壳径：约20 mm)摄食行为、摄食量和生长的影响,以评估不同流速下,海胆摄食行为和生长的差异。实验结果表明,流速对光棘球海胆的存活无显著影响,但显著影响其生长。2 cm/s下海胆的壳径和体重显著大于10和20 cm/s。在实验开始后的第2周和第3周,2 cm/s下海胆的体重和壳径已显著高于20 cm/s。流速显著影响光棘球海胆的摄食量(P<0.001)和觅食行为(20 cm/s,P=0.004),但口器咬合行为未受显著影响(P=0.113)。管足附着时间在流速为10 cm/s和20 cm/s下显著长于其在2 cm/s。同样的,相较于2 cm/s(P=0.02)和10 cm/s(P=0.03),20 cm/s的流速可显著削弱光棘球海胆的翻正行为。综上,高流速(20 cm/s)通过影响海胆管足活动削弱其觅食行为(而非摄食行为),进而降低其...     

Effects of body lipid content on the resting metabolic rate and postprandial metabolic response in the southern catfish Silurus meridionalis

We assessed the effects of body lipid content on the resting metabolic rate and specific dynamic action (SDA) of the southern catfish Silurus meridionalis. Obese and lean fish were obtained by feeding the fish with two different feeds at 27.5 °C for 4 weeks prior to the experiment. The fish were fed with experimental diets with a meal size of 4% by body mass. A continuous-flow respirometer was used to determine the oxygen consumption rate at 2-h intervals until the postprandial oxygen consumption rate had returned to the preprandial level. The body lipid content of the obese fish was significantly greater than that of the lean fish. The metabolic parameters evaluated (resting metabolic rate, peak metabolic rate (R_peak), factorial ratio, time to peak, duration, energy expended on SDA (SDA_E), or SDA coefficient) were not significantly affected by body fat content in terms of the whole-body or mass-specific values. Increased body fat content did not decrease the resting metabolic rate in the southern catfish, which might be due to the higher levels of highly unsaturated fatty acids in these fish. The results also suggest that the body composition does not appear to affect the SDA response.     

Influence of hypoxia on cardiac functions in the grass shrimp (Palaemonetes pugio Holthuis)

Crustaceans frequently encounter hypoxic water and have evolved a variety of compensatory mechanisms to deal with low O₂ conditions. Typically, large decapod crustaceans attempt to maintain cardiac output by increasing stroke volume to compensate for the hypoxia-induced bradycardia. Grass shrimp (Palaemonetes pugio), small hypoxic tolerant decapod crustaceans, were used to investigate cardiac responses to hypoxia in a smaller crustacean using videomicroscopy and dimensional analysis techniques. In addition, these techniques were compared to the more established dye dilution technique for calculation of cardiac output. No significant difference was found between the two methods for determining cardiac output in grass shrimp. Cardiac parameters (heart rate f_H, stroke volume V_S, and cardiac output V_b) were monitored in grass shrimp exposed to progressive hypoxia (P_O2s=20, 13.3, 10, 5.3, and 2 KPa O₂). Shrimp exhibit a cardiac response to hypoxia that is atypical when compared to larger crustaceans. Cardiac output was maintained until water P_O2 fell below 10 KPa O₂. This maintenance of V_b is consistent in both large and small decapods, however the mechanism differs. In grass shrimp, V_S was P_O2 dependent and declined significantly while f_H increased significantly when P_O2 was reduced to 13.3 KPa O₂.     

Effects of acclimation and acute temperature change on specific dynamic action and gastric processing in the green shore crab, Carcinus maenas

The effects of temperature acclimation and acute temperature change were investigated in postprandial green shore crabs, Carcinus maenas. Oxygen uptake, gut contractions and transit rates and digestive efficiencies were measured for crabs acclimated to either 10 °C or 20 °C and subsequently exposed to treatment temperatures of 5, 15, or 25 °C. Temperature acclimation resulted in a partial metabolic compensation in unfed crabs, with higher oxygen uptake rates measured for the 10 °C acclimated group exposed to acute test temperatures. The Q₁₀ values were higher than normal, probably because the acute temperature change prevented crabs from fully adjusting to the new temperature. Both the acclimation and treatment temperature altered the characteristics of the specific dynamic action (SDA). The duration of the response was longer for 20 °C acclimated crabs and was inversely related to the treatment temperature. The scope (peak oxygen consumption) was also higher for 20 °C acclimated crabs with a trend towards an inverse relationship with treatment temperature. Since the overall SDA (energy expenditure) is a function of both duration and scope, it was also higher for 20 °C acclimated crabs, with the highest value measured at the treatment temperature of 15 °C. The decline in total SDA after acute exposure to 5 and 25 °C suggests that both cold stress and limitations to oxygen supply at the temperature extremes could be affecting the SDA response. The contractions of the pyloric sac of the foregut region function to propel digesta through the gut, and contraction rates increased with increasing treatment temperature. This translated into faster transit rates with increasing treatment temperatures. Although pyloric sac contractions were higher for 20 °C acclimated crabs, temperature acclimation had no effect on transit rates. This suggests that a threshold level in pyloric sac contraction rates needs to be reached before it manifests itself on transit rates. Although there was a correlation between faster transit times and the shorter duration of the SDA response with increasing treatment temperature, transit rates do not make a good proxy for calculating the SDA characteristics. The digestive efficiency showed a trend towards a decreasing efficiency with increasing treatment temperature; the slower transit rates at the lower treatment temperatures allowing for more efficient nutrient absorption. Even though metabolic rates of 10 °C acclimated crabs were higher, there was no effect of acclimation temperature on digestive efficiency. This probably occurred because intracellular enzymes and digestive enzymes are modulated through different control pathways. These results give an insight into the metabolic and digestive physiology of Carcinus maenas as it makes feeding excursions between the subtidal and intertidal zones.     

Physiological changes in the Norway lobster Nephrops norvegicus (L.) escaping and discarded from commercial trawls on the West Coast of Scotland: II. Disturbances in haemolymph respiratory gases, tissue metabolites and swimming performance after capture and during recovery

The physiological states of trawled and creel-caught (control) Norway Lobsters (Nephrops norvegicus (L.)) captured on grounds off the West Coast of Scotland (120–150 m) were compared. Undersized “discards” (<25–35 mm carapace length) were sampled directly from the cod-end and following recovery in underwater (u/w) cages at a mean depth of 24 m. “Escaped” animals which had passed through the cod-end meshes and collected in a small-meshed net “cover” were transferred without emersion (air-exposure) for sampling on-board. Some of these individuals were also transferred by SCUBA divers to u/w cages. Haemolymph PO₂, PCO₂ and pH measurements showed that both discarded and escaped animals experienced moderate internal hypoxia, hypercapnia and acidosis which became severe after 1 h emersion of the former on deck. A marked handling effect was evident in which haemolymph PO₂ declined and PCO₂ became elevated in both controls and recovering trawled animals. In both discarded and escaped animals haemolymph l-lactate and d-glucose concentrations were high compared to controls, but with levels significantly lower in escapes suggesting less tail-flip swimming activity within the cod-end cover. Further emersion had little effect on haemolymph l-lactate in discards. Recovery to control levels of both metabolites occurred within 24 h in u/w cages but the exercising of captured individuals (by tactile stimulation) produced further significant increases. Abdominal flexor muscle concentrations of l-lactate were also elevated in discards and escapes and positively correlated with haemolymph levels. Muscle glycogen showed severe depletion in both groups compared to unexercised controls and 1 h emersion reduced levels drastically (to 20% of normal concentrations). High haemolymph ammonia (T_amm) was characteristic of both trawled groups but was reduced rapidly during recovery. These metabolite changes were accompanied by reductions in the number of escape swimming tail-flips that could be elicited before exhaustion, particularly in discards. This reduction in performance was evident in discards even after 24 h recovery, but escapes showed almost normal responses. The severity of the physiological stresses experienced during trawling, and to a lesser extent in escaped animals, and their effects on recovery of undersized discards regaining the seabed, is discussed. These findings may help us predict the survival, longer-term recovery and fitness of fished N. norvegicus, and their potential contribution to the regeneration of exploited populations.