Arjan P. Palstra

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 60xa0g 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., 4xa0ng 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.

PCBs and the energy cost of migration in the European eel (Anguilla anguilla L.)

The effect of polychlorinated biphenyls (PCBs) on the energy consumption of fasting silver European eel (Anguilla anguilla L.) was studied over a 27-day period during which the animals were at rest or were swimming 800 km in Blazka swim tunnels. Three-year-old female hatchery eels (silver stage) between 73 and 80 cm long weighing around 1 kg were dosed intraperitoneally with PCBs at a nominal dosage of 10x the consumption standard as a mixture representative for planar (7 microg PCB126/kg eel), non-planar (5 mg PCB153/kg eel) and metabolizable PCBs (50 microg PCB77/kg eel) found in wild eel, or only with the vehicle (corn oil, 10 ml/kg eel). Four major observations were made: (1) PCB-exposed animals lose less weight compared to their unexposed controls; (2) PCB-concentrations on a lipid basis are 2.8-14 times higher in swimming compared to resting animals; (3) the standard metabolic rate is significantly lower in the PCB-exposed animals than in unexposed controls. In addition, PCB-exposure significantly reduces oxygen consumption during swimming, and starting at 400 km (18 days) this effect increases with time; (4) the relative spleen and liver weight significantly increased in the PCB-swim animals but not in the PCB-rest animals. The swimming animals lost about 75% more weight compared to resting animals and had about 50% lower plasma fat content. Hematocrit, haemoglobin, plasma pH, ion levels (sodium and potassium), and plasma lactate were not affected by PCB-exposure or swimming. Apparently, the current levels of PCBs and other dioxin-like compounds may seriously impair the reproduction of the European eel.

Fish under exercise

Improved knowledge on the swimming physiology of fish and its application to fisheries science and aquaculture (i.e., farming a fitter fish) is currently needed in the face of global environmental changes, high fishing pressures, increased aquaculture production as well as increased concern on fish well-being. Here, we review existing data on teleost fish that indicate that sustained exercise at optimal speeds enhances muscle growth and has consequences for flesh quality. Potential added benefits of sustained exercise may be delay of ovarian development and stimulation of immune status. Exercise could represent a natural, noninvasive, and economical approach to improve growth, flesh quality as well as welfare of aquacultured fish: a FitFish for a healthy consumer. All these issues are important for setting directions for policy decisions and future studies in this area. For this purpose, the FitFish workshop on the Swimming Physiology of Fish (http://www.ub.edu/fitfish2010) was organized to bring together a multidisciplinary group of scientists using exercise models, industrial partners, and policy makers. Sixteen international experts from Europe, North America, and Japan were invited to present their work and view on migration of fishes in their natural environment, beneficial effects of exercise, and applications for sustainable aquaculture. Eighty-eight participants from 19 different countries contributed through a poster session and round table discussion. Eight papers from invited speakers at the workshop have been contributed to this special issue on The Swimming Physiology of Fish.

Establishing zebrafish as a novel exercise model: swimming economy, swimming-enhanced growth and muscle growth marker gene expression.

Background Zebrafish has been largely accepted as a vertebrate multidisciplinary model but its usefulness as a model for exercise physiology has been hampered by the scarce knowledge on its swimming economy, optimal swimming speeds and cost of transport. Therefore, we have performed individual and group-wise swimming experiments to quantify swimming economy and to demonstrate the exercise effects on growth in adult zebrafish. Methodology/Principal Findings Individual zebrafish (nu200a=u200a10) were able to swim at a critical swimming speed (Ucrit) of 0.548±0.007 m s−1 or 18.0 standard body lengths (BL) s−1. The optimal swimming speed (Uopt) at which energetic efficiency is highest was 0.396±0.019 m s−1 (13.0 BL s−1) corresponding to 72.26±0.29% of Ucrit. The cost of transport at optimal swimming speed (COTopt) was 25.23±4.03 µmol g−1 m−1. A group-wise experiment was conducted with zebrafish (nu200a=u200a83) swimming at Uopt for 6 h day−1 for 5 days week−1 for 4 weeks vs. zebrafish (nu200a=u200a84) that rested during this period. Swimming zebrafish increased their total body length by 5.6% and body weight by 41.1% as compared to resting fish. For the first time, a highly significant exercise-induced growth is demonstrated in adult zebrafish. Expression analysis of a set of muscle growth marker genes revealed clear regulatory roles in relation to swimming-enhanced growth for genes such as growth hormone receptor b (ghrb), insulin-like growth factor 1 receptor a (igf1ra), troponin C (stnnc), slow myosin heavy chain 1 (smyhc1), troponin I2 (tnni2), myosin heavy polypeptide 2 (myhz2) and myostatin (mstnb). Conclusions/Significance From the results of our study we can conclude that zebrafish can be used as an exercise model for enhanced growth, with implications in basic, biomedical and applied sciences, such as aquaculture.

Swimming physiology of European silver eels (Anguilla anguilla L.): energetic costs and effects on sexual maturation and reproduction

The European eel migrates 5,000–6,000xa0km to the Sargasso Sea to reproduce. Because they venture into the ocean in a pre-pubertal state and reproduce after swimming for months, a strong interaction between swimming and sexual maturation is expected. Many swimming trials have been performed in 22 swim tunnels to elucidate their performance and the impact on maturation. European eels are able to swim long distances at a cost of 10–12xa0mg fat/km which is 4–6 times more efficient than salmonids. The total energy costs of reproduction correspond to 67% of the fat stores. During long distance swimming, the body composition stays the same showing that energy consumption calculations cannot be based on fat alone but need to be compensated for protein oxidation. The optimal swimming speed is 0.61–0.67xa0mxa0s−1, which is ~60% higher than the generally assumed cruise speed of 0.4xa0mxa0s−1 and implies that female eels may reach the Sargasso Sea within 3.5xa0months instead of the assumed 6xa0months. Swimming trials showed lipid deposition and oocyte growth, which are the first steps of sexual maturation. To investigate effects of oceanic migration on maturation, we simulated group-wise migration in a large swim-gutter with seawater. These trials showed suppressed gonadotropin expression and vitellogenesis in females, while in contrast continued sexual maturation was observed in silver males. The induction of lipid deposition in the oocytes and the inhibition of vitellogenesis by swimming in females suggest a natural sequence of events quite different from artificial maturation protocols.

AMP-Activated Protein Kinase Plays an Important Evolutionary Conserved Role in the Regulation of Glucose Metabolism in Fish Skeletal Muscle Cells

AMPK, a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AMPK is activated by changes in the intracellular AMP∶ATP ratio when ATP consumption is stimulated by contractile activity but also by AICAR and metformin, compounds that increase glucose transport in mammalian muscle cells. However, the possible role of AMPK in the regulation of glucose metabolism in skeletal muscle has not been investigated in other vertebrates, including fish. In this study, we investigated the effects of AMPK activators on glucose uptake, AMPK activity, cell surface levels of trout GLUT4 and expression of GLUT1 and GLUT4 as well as the expression of enzymes regulating glucose disposal and PGC1α in trout myotubes derived from a primary muscle cell culture. We show that AICAR and metformin significantly stimulated glucose uptake (1.6 and 1.3 fold, respectively) and that Compound C completely abrogated the stimulatory effects of the AMPK activators on glucose uptake. The combination of insulin and AMPK activators did not result in additive nor synergistic effects on glucose uptake. Moreover, exposure of trout myotubes to AICAR and metformin resulted in an increase in AMPK activity (3.8 and 3 fold, respectively). We also provide evidence suggesting that stimulation of glucose uptake by AMPK activators in trout myotubes may take place, at least in part, by increasing the cell surface and mRNA levels of trout GLUT4. Finally, AICAR increased the mRNA levels of genes involved in glucose disposal (hexokinase, 6-phosphofructokinase, pyruvate kinase and citrate synthase) and mitochondrial biogenesis (PGC-1α) and did not affect glycogen content or glycogen synthase mRNA levels in trout myotubes. Therefore, we provide evidence, for the first time in non-mammalian vertebrates, suggesting a potentially important role of AMPK in stimulating glucose uptake and utilization in the skeletal muscle of fish.

Deep RNA sequencing of the skeletal muscle transcriptome in swimming fish.

Deep RNA sequencing (RNA-seq) was performed to provide an in-depth view of the transcriptome of red and white skeletal muscle of exercised and non-exercised rainbow trout (Oncorhynchus mykiss) with the specific objective to identify expressed genes and quantify the transcriptomic effects of swimming-induced exercise. Pubertal autumn-spawning seawater-raised female rainbow trout were rested (nu200a=u200a10) or swum (nu200a=u200a10) for 1176 km at 0.75 body-lengths per second in a 6,000-L swim-flume under reproductive conditions for 40 days. Red and white muscle RNA of exercised and non-exercised fish (4 lanes) was sequenced and resulted in 15–17 million reads per lane that, after de novo assembly, yielded 149,159 red and 118,572 white muscle contigs. Most contigs were annotated using an iterative homology search strategy against salmonid ESTs, the zebrafish Danio rerio genome and general Metazoan genes. When selecting for large contigs (>500 nucleotides), a number of novel rainbow trout gene sequences were identified in this study: 1,085 and 1,228 novel gene sequences for red and white muscle, respectively, which included a number of important molecules for skeletal muscle function. Transcriptomic analysis revealed that sustained swimming increased transcriptional activity in skeletal muscle and specifically an up-regulation of genes involved in muscle growth and developmental processes in white muscle. The unique collection of transcripts will contribute to our understanding of red and white muscle physiology, specifically during the long-term reproductive migration of salmonids.

Temporal expression of hepatic estrogen receptor 1, vitellogenin1 and vitellogenin2 in European silver eels

Because European silver eels have never been caught during or after their 6000-km reproductive migration to the Sargasso Sea, all existing knowledge on their sexual maturation comes from hormonal stimulation. Silver eels that start their oceanic migration are still immature with pre-vitellogenic oocytes. Hence we assumed that vitellogenesis should start with the expression of the estrogen receptor in the liver before the circulating 17beta-estradiol (E2) can have any effect. In this study we followed the hepatic vitellogenesis upon 4 weekly injections with carp pituitary extracts (CPE). New molecular primers for the expression of the estrogen receptor 1 (esr1), vitellogenin1 (vtg1) and vitellogenin2 (vtg2) in the liver were developed. Sequences of vtg2 and esr1 were not previously described in Anguilla anguilla. All eels showed weekly increase of the eye size and pectoral fin length, which are signs of early maturation. The same occurred with the gonadosomatic index, the oocyte stage and diameter, and number of deposited fat droplets. Early vitellogenesis appeared as a 3-step process (1) E2-levels and esr1 expression were significantly increased already after one injection, (2) vtg1 and vtg2 expression were significantly increased after one and two injections, respectively, and (3) vtg1 and vtg2 expression increased further after three and four injections. Then also plasma calcium (corresponds with plasma vitellogenin) increased and yolk globuli appeared in the oocytes. These results show that esr1 is the first of the three genes examined that is expressed during the onset of hepatic vitellogenesis. Furthermore, ovarian vitellogenesis (appearance of yolk globuli in oocytes) occurs 1-2 weeks later than the onset of hepatic vitellogenesis.

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 fshβ and lhβ expression, plasma 17β-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 fshβ, lhβ, 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 fshβ expression and E2 profile in vitellogenic hormonally matured eel females that were more similar to the profile observed in other naturally maturing fish.

First artificial hybrid of the eel species Anguilla australis and Anguilla anguilla

BackgroundStudies on artificial hybridization of different Anguilla species were conducted recently, i.e. female A. australis with male A. dieffenbachii, and female A. japonica with male A. anguilla. The existence of these artificial hybrids was however not demonstrated by independent genetic methods. Two species - A. anguilla and A. australis - that are phylogenetically close but have different sexual maturation times (12-25 weeks and 6-8 weeks, respectively), were expected to produce favourable hybrids for reproduction studies.ResultsA modification of the protocol for the reproduction of Anguilla japonica was used to produce eight-day Anguilla australis larvae, with a success rate of 71.4%. Thus ten out of 14 females produced eggs that could be fertilized, and three batches resulted in mass hatching. Hybrid larvae from female A. australis x male A. Anguilla survived for up to seven days post fertilization (dpf). The early development of the hybrid showed typical characteristics of A. anguilla tail pigmentation at 50 hours post fertilization (hpf), indicating expression of genes derived from the father.ConclusionsIn this paper we describe the first production of hybrid larvae from male A. anguilla and female A. australis and their survival for up to 7 dpf. A species-specific nucleotide difference in the 18 S rDNA gene confirmed that genes from both A. australis and A. anguilla were present in the hybrids. The developmental stages of the hybrid eel embryos and larvae are described using high resolution images. Video footage also indicated a heart beat in 5-dpf larva.