The Anguilla spp. migration problem: 40 million years of evolution and two millennia of speculation.

Anguillid eels Anguilla spp. evolved between 20 and 40 million years ago and possess a number of remarkable migratory traits that have fascinated scientists for millennia. Despite centuries of effort, the spawning areas and migrations are known only for a few species. Even for these species, information on migratory behaviour is remarkably sketchy. The latest knowledge on the requirements for successful migration and field data on the migrations of adults and larvae are presented, how experiments on swimming efficiency have progressed the understanding of migration are highlighted and the challenges of swimming at depth considered. The decline of Anguilla spp. across the world is an ongoing concern for fisheries and environmental managers. New developments in the knowledge of eel migration will, in addition to solving a centuries old mystery, probably help to identify how this decline might be halted or even reversed.

Quantitative bioassays for measuring biologically functional gonadotropins based on eel gonadotropic receptors.

Significant declines in eel stocks have been noted in many parts of the world. Because eel aquaculture is dependent on wild-caught juveniles, there is a need to achieve artificial reproduction. Adult eel maturation is currently induced by repeated injections of purified gonadotropin (human chorionic gonadotropin [hCG]) or pituitary extract. Thus the determination of the biological efficacy and quantification of internal levels of gonadotropic hormones is important for optimizing artificial reproduction protocols. To quantify the plasma levels of biologically functional gonadotropic hormones, we developed a bioassay for luteinizing hormone (LH) and follicle-stimulating hormone (FSH) based on the stable expression of receptors in HEK293 cells of the Japanese eel Anguilla japonica LH (ajLHR) and the European eel Anguilla anguilla FSH (aaFSHR), respectively. Such cells also contain a firefly luciferase reporter gene driven by a cAMP-responsive element (CRE-Luc). We found that the obtained stable cells, with ajLHR, responded linearly to a more than 100,000-fold concentration range of hCG diluted in saline. The cells with aaFSHR showed a linear response to a 1000-fold concentration range of salmon pituitary extract mixed with saline. The biological functionality of the LH and FSH bioassays was validated using hCG, human FSH, and pituitary extracts from salmon, carp and eel. Since the toxins in eel plasma damaged the HEK293 cells, the protocol was adapted to selectively inactivate the toxins by heating at 37°C for 24h. This process successfully enabled the monitoring of hormone levels in blood plasma sampled from hCG-injected eels. In this paper, we describe the development of gonadotropin bioassays that will be useful for improving reproduction protocols in eel aquaculture.

Influence of temperature regime on endocrine parameters and vitellogenesis during experimental maturation of European eel (Anguilla anguilla) females.

We examined the effect of temperature in European silver eels during their maturation induced by injections of carp pituitary extract on endocrine parameters: pituitary fshß and lhß expression, plasma 17ß-estradiol (E2) and vitellogenin, estrogen receptor 1 (esr1), and vitellogenin 2 (vtg2) expression in liver. A variable thermal regime (T10) that increased from 10° to 14° and 17°C was compared with a constant 20°C regime (T20) during 12 weeks. T10 caused a faster development until week 8, higher fshß, lhß, esr1 expression, and higher E2 levels. The results strongly suggest that T10 is inducing a higher endogenous FSH level which increases the E2 circulating level during vitellogenesis. A variable thermal regime induced an fshß expression and E2 profile in vitellogenic hormonally matured eel females that were more similar to the profile observed in other naturally maturing fish.

Multiple strategies of Lake Victoria cichlids to cope with lifelong hypoxia include hemoglobin switching.

Many fish species adapt to hypoxia by reducing their metabolic rate and increasing hemoglobin-oxygen (Hb-O(2)) affinity. Pilot studies with young broods of cichlids showed that the young could survive severe hypoxia in contrast with the adults. It was therefore hypothesized that early exposure results in improved oxygen transport. This hypothesis was tested using split brood experiments. Broods of Astatoreochromis alluaudi, Haplochromis ishmaeli, and a tilapia hybrid (Oreochromis) were raised either under normoxia (NR; 80-90% air saturation) or hypoxia (HR; 10% air saturation). The activity of the mitochondrial citrate synthase was not different between NR and HR tilapia, but was significantly decreased in HR A. alluaudi and H. ishmaeli, indicating lowered maximum aerobic capacities. On the other hand, hemoglobin and hematocrit levels were significantly higher in all HR fish of the three species, reflecting a physiological adaptation to safeguard oxygen transport capacity. In HR tilapia, intraerythrocytic GTP levels were decreased, suggesting an adaptive increase of blood-O(2) affinity. Similar changes were not found in HR H. ishmaeli. In this species, however, all HR specimens exhibited a distinctly different iso-Hb pattern compared with their NR siblings, which correlated with a higher intrinsic Hb-O(2) affinity in the former. All HR cichlids thus reveal left-shifted Hb-O(2) equilibrium curves, mediated by either decreased allosteric interaction or, in H. ishmaeli, by the production of new hemoglobins. It is concluded that the adaptation to lifelong hypoxia is mainly due to improved oxygen transport.

Does a 5500-km swim trial stimulate early sexual maturation in the European eel (Anguilla anguilla L.)?

The catadromous European eel (Anguilla anguilla L.) undertakes a 6000-km spawning migration from its freshwater habitats to the Sargasso Sea. In large Blazka swim tunnels of 127 l, the physiological effect of such a prolonged swimming performance on sexual maturation in adult female eels was investigated. Two groups of eels were placed in swim tunnels for 173 days, one group was able to swim at 0.5 body lengths/second (Swim group) covering a distance of c. 5500-km over the experimental period, and one group kept in static (End Control group). A control group was sampled at the start of the experiment in order to determine the initial stage of reproductive development (Initial Control group). At the end of the swim trial, the maturation parameters 11-ketotestosterone, pituitary levels of LH and plasma levels of estradiol were higher (although not significantly) in the Swim compared to the End Control group. In addition, no significant differences were observed in most measured morphometric and reproductive parameters, including eye-index, gonadosomatic index, hepatosomatic index, and plasma levels of vitellogenin, cortisol and melanophore-stimulating hormone (MSH). Also, pituitary levels of both MSH, and adrenocorticotropic hormone (ACTH) were unaffected. In contrast, the oocyte diameter was found to be significantly higher in the Swim compared to the End Control group. Based on these observations we conclude that a period of prolonged swimming might be a physiological stimulus necessary for the onset of maturation in the European eel.

Are dioxin-like contaminants responsible for the eel (Anguilla anguilla) drama?

Eel populations worldwide are dangerously close to collapsing. Our study is the first to show that current levels of dioxin-like contaminants are strong candidates because of their devastating effects on development and survival of eel embryos. Female and male silver eels were artificially stimulated to maturation and reproduction by treatment with carp pituitary extracts and hCG, respectively. During maturation of female European silver eels, about 60 g fat per kg eel is incorporated in the oocytes. Together with the fat, however, persistent organic pollutants such as dioxin-like polychlorinated biphenyls (PCBs) are incorporated too. The total dioxin-like toxic potency of the individual gonad batches was determined as 2,3,7,8-tetrachlorodibenzo-p-dioxine equivalents (TEQs), using an in vitro reporter gene assay. The observed differences in development and survival showed a significant negative correlation with the TEQ levels in the gonads, already at levels far below the maximal allowable level for fish consumption, i.e., 4 ng TEQ/kg fish. The clear inverse relationship between the TEQ level and the survival period of the fertilised eggs strongly suggests that the current levels of dioxin-like compounds seriously impair the reproduction of the European eel. The peak of the environmental levels of dioxin-like PCBs and the decline of eel coincide worldwide, further suggesting that, in addition to other threats, these contaminants contributed significantly to the current collapse in eel populations.

The adrenergic control of hepatic glucose and FFA metabolism in rainbow trout (Oncorhynchus mykiss): increased sensitivity to adrenergic stimulation with fasting.

The adrenergic control of glucose and FFA release was studied in hepatocytes of rainbow trout (Oncorhynchus mykiss), which were either normally fed or fasted for 3 weeks. Isolated hepatocytes were incubated with adrenaline, noradrenaline, or isoprenaline (nonselective beta-agonist). Identification of the hepatic beta-adrenoceptor was combined with quantification of the difference in its affinity for adrenaline and noradrenaline. To identify the beta-adrenoceptor subtype, isoprenaline incubations were combined with atenolol (selective beta(1)-antagonist) or ICI 118,551 (selective beta2-antagonist). Stimulation of the beta-adrenoceptor resulted in mobilisation of glucose, which was inhibited by ICI 118,551 thus pointing to a beta2-subtype. The affinity of the beta2-adrenoceptor for isoprenaline and adrenaline (beta2-values of 8.3 and 7.9) was clearly higher than for noradrenaline (beta2-value of 6.5). This indicates that at physiological concentrations beta2-adrenoceptors in trout are mainly stimulated by adrenaline and not by noradrenaline. A significant effect of beta-adrenoceptor stimulation on the FFA release was also found, although only at high concentrations (i.e., 10(-6) and 10(-5)M). Again the beta2-adrenoceptor appeared to mediate the stimulation of hepatic FFA release. Upon fasting, both the basal glucose and FFA release were strongly decreased. The ratio between glucose and FFA release decreased from 15.4 to 4.3 upon fasting and at this ratio the energy output for both metabolites became equal. The mobilisation of FFA upon adrenergic stimulation was relatively conserved, namely -35% upon fasting, as opposed to -89% in mobilisation of glucose. This indicates that upon fasting FFA gain importance in hepatic metabolism. The hepatic sensitivity to adrenergic stimulation is enhanced upon fasting, as indicated by an increased beta2-value from 8.3 to 8.9.

Free fatty acid metabolism in the air-breathing African catfish (Clarias gariepinus) during asphyxia.

In several waterbreathing fish species, hypoxia induces a decrease in plasma free fatty acid (FFA) levels as opposed to an increase in air-breathing mammals. We hypothesised that this change is coupled to the mode of breathing. Therefore, we followed the metabolic response of cannulated air-breathing African catfish to an 8-h asphyxia period. The hematocrit and hemoglobin increased significantly upon asphyxia. However, no change was observed in the mean cellular hemoglobin concentration, indicating that more erythrocytes were brought into circulation. A continuous increase in plasma lactate concentration during asphyxia showed permanent activation of anaerobic glycolysis, pointing to a persistent oxygen shortage. Plasma glucose levels did not change, but FFA levels decreased significantly upon asphyxia with a concomitant increase in plasma noradrenaline levels. Thus, these results suggest that in the air-breathing African catfish noradrenaline mediated a decrease in plasma FFA levels similar to that in waterbreathing fish species.

Hematology patterns of migrating European eels and the role of EVEX virus.

We show that European eels infected with the rhabdovirus EVEX (Eel Virus European X) virus, developed hemorrhage and anemia during simulated migration in large swim tunnels, and died after 1000-1500 km. In contrast, virus-negative animals swam 5500 km, the estimated distance to the spawning ground of the European eel in the Sargasso Sea. Virus-positive eels showed a decline in hematocrit, which was related to the swim distance. Virus-negative eels showed a slightly increased hematocrit. Observed changes in plasma lactate dehydrogenase (LDH), total protein and aspartate aminotransferase (AAT) are indicative of a serious viral infection. Based on these observations, we conclude that eel virus infections may adversely affect the spawning migration of eels, and could be a contributing factor to the worldwide decline of eel.

Nutritional and hormonal control of lipolysis in isolated gilthead seabream (Sparus aurata) adipocytes.

We examined the effects of diet composition and fasting on lipolysis of freshly isolated adipocytes from gilthead seabream (Sparus aurata). We also analyzed the effects of insulin, glucagon, and growth hormone (GH) in adipocytes isolated from fish fed with different diets. Basal lipolysis, measured as glycerol release, increased proportionally with cell concentration and time of incubation, which validates the suitability of these cell preparations for the study of hormonal regulation of this metabolic process. Gilthead seabream were fed two different diets, FM (100% of fish meal) and PP (100% of plant protein supplied by plant sources) for 6 wk. After this period, each diet group was divided into two groups: fed and fasted (for 11 days). Lipolysis was significantly higher in adipocytes from PP-fed fish than in adipocytes from FM-fed fish. Fasting provoked a significant increase in the lipolytic rate, about threefold in isolated adipocytes regardless of nutritional history. Hormone effects were similar in the different groups: glucagon increased the lipolytic rate, whereas insulin had almost no effect. GH was clearly lipolytic, although the relative increase in glycerol over control was lower in isolated adipocytes from fasted fish compared with fed fish. Together, we demonstrate for the first time that lipolysis, measured in isolated seabream adipocytes, is affected by the nutritional state of the fish. Furthermore, our data suggest that glucagon and especially GH play a major role in the control of adipocyte lipolysis.

Differential role of adrenoceptors in control of plasma glucose and fatty acids in carp, Cyprinus carpio (L.).

Carp were cannulated in the dorsal aorta, and after 2 days of recovery they were infused with 1) norepinephrine, 2) yohimbine (alpha(2)-antagonist) plus norepinephrine, 3) clonidine (alpha(2)-agonist), and 4) isoproterenol (nonselective beta-agonist). Norepinephrine lowered the plasma free fatty acid (FFA) level and raised the plasma glucose level for several hours. Norepinephrine in combination with the alpha(2)-antagonist yohimbine resulted in retardation of the FFA decrease, indicating the involvement of alpha(2)-adrenoceptors. Infusion with the partial alpha(2)-agonist clonidine had a smaller effect. Infusion with isoproterenol caused a marked increase of glucose levels, and unexpectedly a decline of plasma FFA levels, indicating a direct involvement of beta-adrenoceptors. Combination of isoproterenol with either atenolol (beta(1)-antagonist) or ICI-118,551 (beta(2)-antagonist) showed that both beta(1)- and beta(2)-adrenoceptors were involved in the glucose release by isoproterenol. Remarkably, the decline of FFA levels was augmented in the presence of ICI-118,551, whereas with atenolol present plasma FFA levels were increased by isoproterenol. Thus it is concluded that in carp both beta(1)- and beta(2)-adrenoceptors mediate glucose release, whereas lipolysis is controlled by inhibitory beta(1)-adrenoceptors and stimulatory beta(2)-adrenoceptors, as well as by inhibitory alpha(2)-adrenoceptors.

Phosphorylation state of red and white muscle in tilapia during graded hypoxia: an in vivo (31)P-NMR study.

The aim of this study was to measure the energetic consequences of hypoxia in different types of skeletal muscle within a single tilapia species (n = 5). To that aim, 81.0 MHz (31)P-nuclear magnetic resonance (NMR) spectra were collected, alternately, from three surface coils placed adjacent to the tissues of interest (dorsal white muscle, ventral white muscle, and lateral red muscle) during a graded hypoxia load over 6 h followed by a 5-h recovery period. The fish were contained in a flow cell, enabling us full control of the oxygen content of the bathing medium. The intracellular pH and the concentrations of ATP, phosphocreatine (PCr), and P(i) were determined from the NMR spectra. For normoxia, biochemical differences for [gamma-ATP], [PCr], and [sugar phosphates] (SP) were observed between all three locations, especially between the red and white muscle. During hypoxia stress, loss of phosphorylated compounds (PCr+P(i)+SP) was observed at all locations but was the most severe in red muscle. When the aerobic (respirometry) and anaerobic ((31)P-NMR) ATP production via an energy balance are compared, flexible metabolic depression is demonstrated during anaerobioses. It is concluded that control of the aerobic and anaerobic component of metabolism during metabolic depression is independent of each other.

Synergistic effect of acidification and hypoxia: in vivo 31P-NMR and respirometric study in fishes.

We used 31P nuclear magnetic resonance (31P-NMR) spectroscopy to measure intracellular pH (pHi) and high-energy phosphate levels of white muscle of the fish tilapia (Oreochromis mossambicus) during exposure to the stressors hypoxia and water acidification separately and simultaneously. The protocol for the graded hypoxia load was 100, 40, 30, 20, 10, 5, and 3% air saturation for 1 h at each level. For environmental acidosis the pH of the water was lowered from 7.6 to 4.0 over 6 h, with time of exposure approximately 11 h. All protocols were followed by 6 h of reoxygenation at 20 degrees C. We also measured total oxygen consumption of the animal. The results of this in vivo study revealed that environmental acidification had no effect on oxygen consumption, pHi, or phosphocreatine depletion. Hypoxia caused moderate changes in these parameters and fast and complete recovery during reoxygenation. In contrast, the combination of environmental acidosis and hypoxia resulted in 50% mortality, increased depletion of the phosphocreatine pool, and a retarded recovery of the pHi during reoxygenation compared with the group with hypoxia as a single stressor. The combination of environmental acidification and hypoxia has a more profound effect and works synergistically compared with the conditions imposed separately. To investigate whether an adaptation response occurred during chronic exposure to environmental acidification, animals were exposed for 6 wk to pH 4.0 before experimentation. The pHi in the white muscle dropped from 7.2 (control group) to 6.9 during this period, whereas no effect was found in the phosphorylated compounds and oxygen consumption. Therefore, it is concluded that no adaptation response occurs in animals exposed to long-term environmental acidosis.

Nuclear magnetic resonance spectroscopy of living systems: applications in comparative physiology.

The most attractive feature of nuclear magnetic resonance spectroscopy (MRS) is the noninvasive and nondestructive measurement of chemical compounds in intact tissues. MRS already has many applications in comparative physiology, usually based on observation of 31P, since levels of phosphorus compounds indicate tissue energy status and are changed during exercise, fatigue, recovery, hypometabolism, anesthesia, hypoxia, hypercapnia, and osmotic and acid stress. Nuclei other than 31P may also be monitored, such as 1H, 13C, 15N, 19F, or 23Na, and applied in biological research. Particularly, 13C-MRS is interesting because it allows the analysis of metabolic pathways in living systems. Applications of MRS in comparative physiology and biochemistry are comprehensively discussed in this review. The main focus is on anaerobic metabolism during hypoxia, ischemia, and exercise. Species as widely different as slime molds, nematodes, frogs, turtles, and ducks have been studied by 31P-MRS. It is not surprising that striking species differences do occur, but many similarities are also observed. Unique is the occurrence of six different phosphagens with different values of Gibbs free energy in polychete worms The presence of a particular phosphagen may be related to the average oxygen tension within the tissues. Phosphagens and their kinases are also discussed in relation to hypercapnia and acid stress. Other topics discussed in this paper are enzyme kinetics, anesthetics, development and growth, parasitism, and the detection of previously unknown compounds.

Behavioral strategy and the physiological stress response in rainbow trout exposed to severe hypoxia.

In higher vertebrates, two opposite behavioral coping strategies can be distinguished that are associated by a typical neuroendocrine pattern. Little is known about the individual variation in the stress response in lower vertebrates such as teleosts. In the present study, rainbow trout were fitted with an indwelling aortic catheter for repeated blood sampling and exposed to severe hypoxia and subsequent recovery and their behavior was characterized semiquantitatively during hypoxia. Blood levels of catecholamines, cortisol, glucose, FFA, lactate, and electrolytes were measured. About 60% of the fish survived the experiment whereas the others died during the recovery period. Behavioral strategy appeared to be highly related to survival since nonsurviving fish displayed strenuous avoidance behavior involving burst type activity whereas surviving fish did not panic and remained quiet. These behavioral differences were associated with marked differences in plasma catecholamine levels, which were 4- to 5-fold higher in nonsurviving fish as compared to survivors whereas the cortisol response tends to be lower in nonsurviving fish. Plasma lactate levels in nonsurvivors were 4- to 5-fold higher as compared to survivors while a severe hyperkalemia developed during recovery indicating the loss of intracellular homeostasis. The individual differences in behavioral concepts and neuroendocrine activation observed in rainbow trout during stress show great similarity with the active and passive coping strategies distinguished in higher vertebrates and may be determinant for survival during hypoxia.

Effect of arterially infused catecholamines and insulin on plasma glucose and free fatty acids in carp.

Common carp (Cyprinus carpio L.), kept at 20 degrees C, were fitted with an indwelling PE-50 cannula in the dorsal aorta. Hormones dissolved in Ringer saline were arterially infused at a rate of 1 microgram.kg-1.min-1 for epinephrine (Epi), 2 micrograms.kg-1.min-1 for norepinephrine (NE), and 1.33 micrograms.kg-1.min-1 for insulin. INfusion of bovine insulin in carp resulted in a long lasting (24 h) decrease of plasma free fatty acids (FFA; -0.41 +/- 0.06 mM) and glucose levels (-3.14 +/- 0.25 mM) compared with preinfusion levels at t = 0. Both Epi and NE induced a marked hyperglycemia although Epi was more potent (+8.2 +/- 0.9 and +6.9 +/- 0.8 mM, respectively). Plasma FFA levels increased by 0.25 +/- 0.03 mM compared with preinfusion levels on Epi infusion. In contrast, during NE infusion, plasma FFA levels decreased significantly by -0.21 +/- 0.03 mM. Plasma insulin titers did not significantly change during infusion of NE or Epi. It is concluded that the ratio of NE to Epi is the major factor that determines the effect of catecholamines on plasma FFA levels in carp. These results may explain species-dependent different effects of hypoxia on FFA metabolism in fish.

Fish muscle energy metabolism measured during hypoxia and recovery: an in vivo 31P-NMR study.

Three fish species were exposed to graded hypoxia levels and allowed to recover. Levels of high-energy phosphate compounds in epaxial white muscle were monitored by in vivo 31P nuclear magnetic resonance (NMR) spectroscopy. Furthermore, O2 consumption of the animals was measured. With increasing hypoxia load, metabolic parameters started to change in the following order: phosphocreatine (PCr)-to-Pi ratio (decrease), O2 consumption (decrease), [PCr] (decrease), intracellular pH (pHi; decrease), Pi (increase), free ADP concentration ([ADP]free; increase), [ATP] (decrease). PCr levels fell with the PO2. After each increment, the [PCr] reached a stable plateau value while, in some cases, a recovery was observed. This recovery could be explained because the balance between anaerobic and aerobic metabolism is continuously fluctuating during hypoxia as a consequence of changes in the activity of the fish. Consequently the [ADP]free are fluctuating, resulting in an activation of the creatine kinase reaction and the anaerobic glycolysis. In all three species, anaerobic glycolysis was activated, but in contrast to anoxia exposure, metabolic suppression was absent. The changes of [ADP]free and [H+] (which influences the position of the creatine kinase equilibrium) are species dependent. Species differences observed in the other parameters were small. It is concluded that the pattern of the activation of anaerobic metabolism under deep hypoxia is different from that under anoxia.

Reversed-phase ion-paired HPLC of purine nucleotides from skeletal muscle, heart and brain of the goldfish, Carassius auratus L.--I. Development of the analytical method.

1. A reversed-phase ion-paired liquid chromatographic (HPLC) method was developed to measure AMP, ADP, ATP, IMP, NAD+ and NADP+ levels in white muscle, heart and brain of anoxic goldfish. 2. Mobile phase parameters of the HPLC method (concentrations of buffer, organic modifier and counter-ion, and pH) were varied to establish the optimal conditions for separation of the compounds of interest. 3. The analytical method was evaluated by calculating some relevant chromatographic parameters (reproducibility and linearity). 4. The HPLC method showed sufficient selectivity, high sensitivity and reproducibility, and excellent linearity.

Reversed-phase ion-paired HPLC of purine nucleotides from skeletal muscle, heart and brain of the goldfish, Carassius auratus L.--II. Influence of environmental anoxia on metabolite levels.

1. The type and the amounts of some (di)nucleotides in white skeletal muscle, heart and brain of anoxic goldfish were established with a reversed-phase ion-paired HPLC method. 2. Significant changes in the levels of these metabolites, as compared to controls, were observed. The most consistent change is the increase of IMP and of IMP-load (IMP/ATP + ADP + AMP) in the three tissues during anoxia. 3. Adenylate energy charges are maintained at a high level in the anoxic white muscle and the anoxic heart. Comparison of these results with those from conventional enzymatic methods for the quantification of (di)nucleotides showed, except for IMP, no significant differences.