The impact of dissolved oxygen concentration on the swimming performance and welfare of ballan wrasse (Labrus bergylta Ascanius, 1767).

Sea lice (Lepeophtheirus salmonis [Krøyer, 1838]) are a key issue for salmon aquaculture, contributing to increased mortality for both wild and farmed salmon if no action is taken. Using cleaner fish can be an effective, drug-free treatment method, and ballan wrasse (Labrus bergylta) is a hardy wrasse species that displays cleaning behavior. With concerns about the overharvest of wild ballan wrasse, many companies farm this species, but the optimal ranges of a wide variety of rearing parameters are still unknown. This study investigated the effect of 6-week exposure to four dissolved oxygen (DO) levels (125%, 100%, 85%, and 75% DO saturation as the percentage of air) on ballan wrasse. Survival; growth (specific growth rate, SGR); condition factor (CF); and primary (cortisol), secondary (glucose, lactate, magnesium), and tertiary stress indicators (swimming performance) were investigated. There were no differences in SGR, CF, survival, or cortisol level among the groups at the end of the 6 weeks. There was variation in the magnitude of the cortisol response to an acute stressor at the end of the 6-week period, with the 75% DO treatment exhibiting a 3.3-fold increase in cortisol compared to a 5.2-fold increase in the control group (100%), which could suggest chronic stress. Relative critical swimming speed (RUcrit) was measured to investigate swimming performance once all groups were returned to 100% DO saturation. The 75% RUcrit was lower than the 100% treatment (1.7 ± 0.18 body length [BL]/s compared to 2.5 ± 0.16 BL/s). Overall, these results suggest that DO levels of 75% trigger physiological changes and therefore may negatively affect welfare.

Effect of prolonged feeding of broodstock diet with increased inclusion of essential n-3 fatty acids on maturing and spawning performance in 3-year-old Atlantic salmon (Salmo salar).

Atlantic salmon (Salmo salar) broodstock recruits are normally fed a specialized diet with a higher content of essential nutrients for a limited time period prior to fasting and transfer to freshwater. Typically, this period lasts for about six months, but may vary among producers. Reduced use of marine ingredients in commercial salmon diets during the last decades has affected the content of essential nutrients, such as n-3 long chained polyunsaturated fatty acids (LC-PUFA), minerals and vitamins. Furthermore, to minimize the risk of losses and implement new breeding achievements faster, breeding companies have shortened the production cycle of broodstock from 4 to 3 years, which may affect the number of fish that are large enough to mature. In the present study, we have extended the broodstock feeding period from 6 to 15 months prior to the freshwater transfer giving a higher content of n-3 LC-PUFA (higher inclusion of marine oils) from February to December (Phase 1), and thereafter a diet with a higher energy content to ensure growth towards the spring and maturation (Phase 2). Four sea cages with approximately 80.000 salmon postsmolt, two sea cages with males and two with females, were given a control diet and an experimental diet. Samples were taken in Phase 1 at start (1.7 kg), mid (3.4 kg) and end Phase 1/start of Phase 2 (8.3 kg), and end of Phase 2 (13.4 kg). The fish were thereafter fasted, and selected fish transferred to landbased freshwater tanks where light and temperature were used to manipulate the spawning time of the fish in two groups (early or late). Due to disease in the facility, measures of egg quality and hatching were only obtained from the early group. During the trial and spawning period, biometrical measurements were recorded, and samples of liver, gonad, fillet and red blood cells (RBC) were collected for fatty acid composition and blood plasma for analysis of lipid and health-related parameters. Samples were also collected for gonadal transcriptomic analysis by microarray and qPCR (end Phase 2) and plasma steroids (end Phase 2, mid maturation and spawning). Males fed the test diet had a larger body size compared to the control group at the end of Phase 2, while no differences were observed between dietary groups for the females. Total mortality in the trial was lower in the test group compared to the control, losses were caused mainly by sea lice treatments, loser fish or cardiomyopathy syndrome (CMS). The dietary LC-PUFA levels in the test diet were reflected in the tissues particularly during Phase 1, but only different in the fillet samples and eggs at the end of Phase 2 and at spawning. Plasma sex steroids content increased at mid maturation and showed lower levels of androgens and estrogens in females fed the test diet compared to the control. At the end of Phase 2, transcriptional analysis showed upregulation of steroidogenic enzymes, although not reflected in changes in plasma steroids in Phase 2, indicating changes to come during maturation. The differences in LC-PUFA content in tissues and plasma steroids did not appear to affect fecundity, sperm quality, egg survival or hatching rate, but the test group had larger eggs compared to the control in the early spawner-group. Prolonged feeding of n-3 LC-PUFA to pre-puberty Atlantic salmon broodstock appears to be important for higher survival in challenging sea cage environments and has an effect on sex steroid production that, together with high energy diet during early maturation, cause the test group to produce larger eggs.

Ultrasonic imaging as a means of monitoring gonadal development in lumpfish (Cyclopterus lumpus).

The commercial farming of juvenile lumpfish requires monitoring of gonadal development to achieve synchronized production. Conventional methods such as gonadosomatic index (GSI), sex hormone analyses, gonadal histology, endoscopy, and gene expression analyses are costly, invasive, and often involve sacrificing the fish. We assessed the efficiency of ultrasound as a non-invasive method for monitoring gonadal development in lumpfish. Based on ultrasound observations, we categorized the fish into six stages; F0 to F5 for females and M0 to M5 for males, that represented maturity levels from immature to spent. Importantly, the ultrasound gonadal stages aligned with histological gonadal stages. Additionally, ultrasound stages aligned with profiles of GSI, testosterone (T), 11-ketotestosterone, and 17ß-estradiol throughout gonadal development including the spawning period. Moreover, these parameters exhibited significant positive correlations with each other reflecting their parallel trends during gonadal development. To minimize the frequency of ultrasound usage and fish handling, we established F3 and M3/M4 as arbitrary thresholds for identifying ripe females and males, respectively. By using these thresholds, the need for regular ultrasound monitoring could be reduced during most of the rearing period. Ultrasound proves to be useful and reliable for monitoring gonadal development in lumpfish, enabling synchronized production of juvenile fish.

Intergenerational effects of early life-stage temperature modulation on gene expression and DNA methylation in Atlantic cod (Gadus morhua).

After suffering several collapses, the cod farming industry is now in the process of trying to re-establish itself. We have used material from Norway's National Cod Breeding Program to study how different early life-stage temperature regimes affect DNA methylation and gene expression. Long-term effects were detected by sampling fish several weeks after the end of differential treatments, and offspring from the different exposure groups was also sampled. Many overlapping genes were found between the different exposure groups and generations, coupled with genes associated with differential CpG methylation levels. Genes involved in muscle fibre development, general metabolic processes and formation of deformities were significantly affected, and genes relevant for intergenerational transfer of epigenetic marks were also detected. We believe the use of environmental cues can be a useful strategy for improving the production of Atlantic cod.

Long-Term Influence of PCB- and PBDE-Spiked Microplastic Spheres Fed through Rotifers to Atlantic Cod (Gadus morhua) Larvae.

Omnipresent microplastics (MPs) in marine ecosystems are ingested at all trophic levels and may be a vector for the transfer of persistent organic pollutants (POPs) through the food web. We fed rotifers polyethylene MPs (1-4 µm) spiked with seven congeners of polychlorinated biphenyls (PCBs) and two congeners of polybrominated diphenyl ethers (PBDEs). In turn, these rotifers were fed to cod larvae from 2-30 days post-hatching (dph), while the control groups were fed rotifers without MPs. After 30 dph, all the groups were fed the same feed without MPs. Whole-body larvae were sampled at 30 and 60 dph, and four months later the skin of 10 g juveniles was sampled. The PCBs and PBDEs concentrations were significantly higher in MP larvae compared to the control larvae at 30 dph, but the significance dissipated at 60 dph. Expression of stress-related genes in cod larvae at 30 and 60 dph showed inconclusive minor random effects. The skin of MP juveniles showed disrupted epithelial integrity, fewer club cells and downregulation of a suite of genes involved in immunity, metabolism and the development of skin. Our study showed that POPs were transferred through the food web and accumulated in the larvae, but that the level of pollutants decreased once the exposure was ceased, possibly related to growth dilution. Considering the transcriptomic and histological findings, POPs spiked to MPs and/or MPs themselves may have long-term effects in the skin barrier defense system, immune response and epithelium integrity, which may potentially reduce the robustness and overall fitness of the fish.

Applying genetic technologies to combat infectious diseases in aquaculture.

Disease and parasitism cause major welfare, environmental and economic concerns for global aquaculture. In this review, we examine the status and potential of technologies that exploit genetic variation in host resistance to tackle this problem. We argue that there is an urgent need to improve understanding of the genetic mechanisms involved, leading to the development of tools that can be applied to boost host resistance and reduce the disease burden. We draw on two pressing global disease problems as case studies-sea lice infestations in salmonids and white spot syndrome in shrimp. We review how the latest genetic technologies can be capitalised upon to determine the mechanisms underlying inter- and intra-species variation in pathogen/parasite resistance, and how the derived knowledge could be applied to boost disease resistance using selective breeding, gene editing and/or with targeted feed treatments and vaccines. Gene editing brings novel opportunities, but also implementation and dissemination challenges, and necessitates new protocols to integrate the technology into aquaculture breeding programmes. There is also an ongoing need to minimise risks of disease agents evolving to overcome genetic improvements to host resistance, and insights from epidemiological and evolutionary models of pathogen infestation in wild and cultured host populations are explored. Ethical issues around the different approaches for achieving genetic resistance are discussed. Application of genetic technologies and approaches has potential to improve fundamental knowledge of mechanisms affecting genetic resistance and provide effective pathways for implementation that could lead to more resistant aquaculture stocks, transforming global aquaculture.

The circulating plasma metabolome of Neoparamoeba perurans-infected Atlantic salmon (Salmo salar).

Metabolomics can provide insights into the dynamic small-molecule fluctuations occurring in response to infection and has become a valuable tool in studying the pathophysiology of diseases in recent years. However, its application in fish disease research is limited. Here, we report the circulating plasma metabolome of Atlantic salmon (Salmo salar) experimentally infected with Neoparamoeba perurans-the causative agent of amoebic gill disease (AGD). Plasma samples were collected from fish with varying degrees of infection inferred from an external gross morphological score of gill pathology (i.e., gill score [GS] 1 -- GS3), where a higher GS indicates advanced infection stage. Uninfected fish (GS0) served as the control. Typical pathologies associated with AGD infection, such as hyperplastic lesions and lamellar fusion, were evident in infected gill samples. Plasma metabolites were identified by ultra-performance liquid chromatography coupled with a high-resolution quadrupole-orbitrap mass spectrometer. Identification of compounds were performed at four levels of certainty, where level 1 provided the most accurate compound identity. A total of 900 compounds were detected in the samples of which 143 were annotated at level 3, 68 on level 2b, 74 on level 2a, and 66 on level 1. Versus GS0, GS1 showed the highest number of significantly affected metabolites (104), which decreased with a higher GS. Adrenaline and adenosine were the two Level 1 compounds significantly affected by AGD regardless of GS, with the former increasing and the latter decreasing in infected fish. Hippuric acid significantly increased in GS1 and GS2, while the tryptophan metabolite indole-3-lactic acid decreased in response to the initial stage of infection but returned to basal levels at a higher GS. There were ten significantly affected metabolic pathways: Eight of which were significantly downregulated while two were downregulated in GS1 relative to GS0. The super-pathway of purine nucleotide salvage was enriched both within the upregulated metabolites in GS1vsGS0 and the down-regulated metabolites in GS3vsGS1. This is the first report on the circulating plasma metabolome of AGD infected salmon, and the results show that low infection levels resulted in a more dramatic metabolomic dysregulation than advanced infection stages. The metabolites identified are potential biological markers for the systemic physiological impact of AGD.

Assessing microplastic as a vector for chemical entry into fish larvae using a novel tube-feeding approach.

A tube-feeding model for administering microplastic (MP, Ø = 30 µm) spheres to fish larvae was employed to quantify the uptake of hydrophobic organic contaminants (HOCs) into the larval body through a single administration of MP. Polychlorinated biphenyl-153 (PCB-153) was used as a representative HOC that can be sorbed to MP in the sea. Atlantic herring (Clupea harengus) larvae (34-51 days post-hatching) were selected as the animal model. The herring larvae were tube-fed a single load of up to 200 polystyrene or polyethylene MP spheres spiked with 14C-labelled PCB-153, and the control larvae were tube-fed an isotonic solution without MP. At the time of sampling (24 h post feeding), some larvae had evacuated all MP spheres from the gut, while others still had MP remaining in the gut. In larvae with a significant number of MP spheres still present in the gut, whole-body scintillation counting (including the MP in the gut lumen) showed elevated levels of the tracer compared to those in the control fish larvae. For larvae in which all or almost all MP had been evacuated by the time of sampling, the tracer levels of the whole body were not significantly different compared to those for the control fish larvae. These data indicate that there was no significant transfer of PCB-153 from contaminated MP into fish larvae within a gut-transit time of <24 h. This study suggests that the vector role of MP in HOC uptake and absorption may be minor compared to that of other HOC uptake pathways.

Low Oxygen Stress During Early Development Influences Regulation of Hypoxia-Response Genes in Farmed Atlantic Salmon (Salmo salar).

Survival and growth of developing salmonids are negatively affected by low oxygen levels within gravel nests in natural streams, and hypoxic stress is often experienced by farmed Atlantic salmon (Salmo salar) within hatcheries. Exposure to hypoxia during early development may have long-lasting effects by altering epigenetic marks and gene expression in oxygen regulatory pathways. Here, we examine the transcriptomic response to low dissolved oxygen (DO) in post-hatch salmon reared continuously in 30%, 60% or 100% DO from fertilization until start of feeding. RNA sequencing revealed multiple differentially expressed genes, including oxygen transporting hemoglobin embryonic α subunit (hbae) and EGLN3 family hypoxia-inducible factor 3 (egln3) which regulates the stability of hypoxia inducible factor 1α (HIF-1α). Both hbae and egln3 displayed expression levels inversely correlated to oxygen concentration, and DNA methylation patterns within the egln3 promoter were negatively associated with the transcript levels. These results suggest that epigenetic processes are influenced by low oxygen levels during early development in Atlantic salmon to upregulate hypoxia-response genes.

Genetic background and embryonic temperature affect DNA methylation and expression of myogenin and muscle development in Atlantic salmon (Salmo salar).

The development of ectothermic embryos is strongly affected by incubation temperature, and thermal imprinting of body growth and muscle phenotype has been reported in various teleost fishes. The complex epigenetic regulation of muscle development in vertebrates involves DNA methylation of the myogenin promoter. Body growth is a heritable and highly variable trait among fish populations that allows for local adaptations, but also for selective breeding. Here we studied the epigenetic effects of embryonic temperature and genetic background on body growth, muscle cellularity and myogenin expression in farmed Atlantic salmon (Salmo salar). Eggs from salmon families with either high or low estimated breeding values for body growth, referred to as Fast and Slow genotypes, were incubated at 8°C or 4°C until the embryonic 'eyed-stage' followed by rearing at the production temperature of 8°C. Rearing temperature strongly affected the growth rates, and the 8°C fish were about twice as heavy as the 4°C fish in the order Fast8>Slow8>Fast4>Slow4 prior to seawater transfer. Fast8 was the largest fish also at harvest despite strong growth compensation in the low temperature groups. Larval myogenin expression was approximately 4-6 fold higher in the Fast8 group than in the other groups and was associated with relative low DNA methylation levels, but was positively correlated with the expression levels of the DNA methyltransferase genes dnmt1, dnmt3a and dnmt3b. Juvenile Fast8 fish displayed thicker white muscle fibres than Fast4 fish, while Slow 8 and Slow 4 showed no difference in muscle cellularity. The impact of genetic background on the thermal imprinting of body growth and muscle development in Atlantic salmon suggests that epigenetic variation might play a significant role in the local adaptation to fluctuating temperatures over short evolutionary time.

Glucocorticoid-Induced Attenuation of the Inflammatory Response in Zebrafish.

Glucocorticoids are steroid hormones that are secreted upon stress. Their effects are mediated by the glucocorticoid receptor, which acts as a transcription factor. Because the antiinflammatory activity of glucocorticoids has been well established, they are widely used clinically to treat many inflammatory and immune-related diseases. However, the exact specificity, mechanisms, and level of regulation of different inflammatory pathways have not been fully elucidated. In the present study, a tail fin amputation assay was used in 3-day-old zebrafish larvae to study the immunomodulatory effects of the synthetic glucocorticoid beclomethasone. First, a transcriptome analysis was performed, which showed that upon amputation mainly immune-related genes are regulated. This regulation was inhibited by beclomethasone for 86% of regulated genes. For two immune-related genes, tlr4bb and alox5ap, the amputation-induced increase was not attenuated by beclomethasone. Alox5ap is involved in eicosanoid biosynthesis, but the increase in leukotriene B4 concentration upon amputation was abolished, and lipoxin A4 levels were unaffected by beclomethasone. Furthermore, we studied the migration of neutrophils and macrophages toward the wound site. Our results show that amputation induced migration of both types of leukocytes and that this migration was dependent on de novo protein synthesis. Beclomethasone treatment attenuated the migratory behavior of neutrophils in a glucocorticoid receptor-dependent manner but left the migration of macrophages unaffected. In conclusion, beclomethasone has a dramatic inhibitory effect on the amputation-induced proinflammatory gene regulation, and this is reflected in an inhibition of the neutrophil migration but not the migration of macrophages, which are likely to be involved in inflammation resolution.

Changes in ovarian gene expression profiles and plasma hormone levels in maturing European eel (Anguilla anguilla); Biomarkers for broodstock selection.

Complete sexual maturation of European eels (Anguilla anguilla) in captivity can only be achieved via injections with gonadotropins. For female eels this procedure takes 4-6months and the response ranges from "unresponsive" to final maturation and ovulation. Reproductive success could be significantly increased via early selection of responders based on predictive markers and minimally invasive sampling methods. To get a better understanding of the genetic background of ovarian maturation of the European eel we performed a pilot deep-sequencing transcriptome analysis of ovarian tissue derived from a yellow eel, a prepubertal silver eel and a post-spawning matured eel. Two key players in steroidogenesis were strongly correlated with advanced sexual maturation, namely P450c17 and liver receptor homolog-1, suggesting that blood plasma steroids might qualify as minimally invasive markers for early detection of responders. Since the predictive value of plasma sex steroid levels for final maturation of the European eel had not yet been carefully examined, we performed an extensive artificial maturation trial. Farmed silver eels were treated with pituitary extracts and sampled at multiple time intervals. Expression of steroidogenesis-related genes in ovarian tissue of responding and non-responding eels after four weekly injections with pituitary extract was compared using a custom-built microarray and RNAseq. Increased expression of 17ß-hsd1 was strongly linked to sexual maturation. Blood plasma levels of sex steroids were measured using ELISAs. We show that a 2.5-fold increase in blood-plasma estradiol level after 4 weekly pituitary extract injections is a strong predictor of final sexual maturation of female European eel.

Comparison of swimming capacity and energetics of migratory European eel (Anguilla anguilla) and New Zealand short-finned eel (A. australis).

The spawning migration of the European eel (Anguilla anguilla) can cover more than 6000 km, while that of the New Zealand short-finned eel (A. australis) is assumed to be approximately 3000 km. Since these species are expected to show adaptive traits to such an important lifetime event, we hypothesized differences in swimming capacity and energetics as a response to this adaptation. In an experimental swimming respirometer set-up, critical swimming speed (Ucrit), optimal swimming speed (Uopt), mass specific oxygen consumption rate (MO2), standard metabolic rate (SMR), active metabolic rate at Ucrit (AMRcrit) and at Uopt (AMRopt), the minimum cost of transport at Uopt (COTmin), and the scope for activity, were assessed and compared between the species. With a similar body length and mass, European eels showed ca. 25% higher values for both Ucrit and Uopt, and 23% lower values for COTmin, compared to New Zealand short-finned eels. However, SMR, AMRcrit, AMRopt, and scope for activity did not differ between the species, indicating very similar swimming physiology traits. This study discusses physiological aspects of long distance migration and provides recommendations for (a) swimming respirometry in anguilliform fish, and (b) telemetry research using externally attached pop-up tags.

The effect of drag and attachment site of external tags on swimming eels: experimental quantification and evaluation tool.

Telemetry studies on aquatic animals often use external tags to monitor migration patterns and help to inform conservation effort. However, external tags are known to impair swimming energetics dramatically in a variety of species, including the endangered European eel. Due to their high swimming efficiency, anguilliform swimmers are very susceptibility for added drag. Using an integration of swimming physiology, behaviour and kinematics, we investigated the effect of additional drag and site of externally attached tags on swimming mode and costs. The results show a significant effect of a) attachment site and b) drag on multiple energetic parameters, such as Cost Of Transport (COT), critical swimming speed (Ucrit) and optimal swimming speed (Uopt), possibly due to changes in swimming kinematics. Attachment at 0.125 bl from the tip of the snout is a better choice than at the Centre Of Mass (0.35 bl), as it is the case in current telemetry studies. Quantification of added drag effect on COT and Ucrit show a (limited) correlation, suggesting that the Ucrit test can be used for evaluating external tags for telemetry studies until a certain threshold value. Uopt is not affected by added drag, validating previous findings of telemetry studies. The integrative methodology and the evaluation tool presented here can be used for the design of new studies using external telemetry tags, and the (re-) evaluation of relevant studies on anguilliform swimmers.

Identification of molecular markers in pectoral fin to predict artificial maturation of female European eels (Anguilla anguilla).

The European eel is a critically endangered species that cannot be reproduced in captivity yet. Artificial maturation of female European eels can be achieved via a laborious and expensive procedure, including weekly injections with pituitary extracts for up to 6 months. The success rate is highly variable and a minimally invasive method for early selection of responsive eels would prevent the unnecessary and lengthy treatment of non-responding individuals. Since sexual maturation of European eels is accompanied by morphological changes of the pectoral fin, we examined whether fin could be used to monitor the response to the hormone treatment. Farmed eels were subjected to weekly injections with pituitary extracts and representative groups were sampled at 0 and 14-18 weeks of hormone treatment. Responders and non-responders were identified based on the gonado-somatic index. Transcriptomes of pectoral fin samples obtained at the start and end of the trial were mapped using Illumina RNAseq. Responders showed 384 and non-responders only 54 differentially expressed genes. Highly stringent selection based on minimum expression levels and fold-changes and a manual re-annotation round yielded 23 up-regulated and 21 down-regulated maturation marker genes. The up-regulated markers belong to five categories: proteases, skin/mucus structural proteins, steroid hormone signaling, tyrosine/dopamine metabolism and lipid metabolism. The down-regulated markers are either blood markers or lectin-related genes. In conclusion, pectoral fin transcriptomes are a rich source of indicator markers for monitoring hormone induced sexual maturation of female European eels. In addition, these markers provide important new insight into several fundamental processes in eel biology.

The pituitary gland of the European eel reveals massive expression of genes involved in the melanocortin system.

Hormones secreted from the pituitary gland regulate important processes such as development, growth and metabolism, reproduction, water balance, and body pigmentation. Synthesis and secretion of pituitary hormones are regulated by different factors from the hypothalamus, but also through feedback mechanisms from peripheral organs, and from the pituitary itself. In the European eel extensive attention has been directed towards understanding the different components of the brain-pituitary-gonad axis, but little is known about the regulation of upstream processes in the pituitary gland. In order to gain a broader mechanistic understanding of the eel pituitary gland, we have performed RNA-seq transcriptome profiling of the pituitary of prepubertal female silver eels. RNA-seq reads generated on the Illumina platform were mapped to the recently assembled European eel genome. The most abundant transcript in the eel pituitary codes for pro-opiomelanocortin, the precursor for hormones of the melanocortin system. Several genes putatively involved in downstream processing of pro-opiomelanocortin were manually annotated, and were found to be highly expressed, both by RNA-seq and by qPCR. The melanocortin system, which affects skin color, energy homeostasis and in other teleosts interacts with the reproductive system, has so far received limited attention in eels. However, since up to one third of the silver eel pituitary's mRNA pool encodes pro-opiomelanocortin, our results indicate that control of the melanocortin system is a major function of the eel pituitary.

Male European eels are highly efficient long distance swimmers: effects of endurance swimming on maturation.

European eels (Anguilla anguilla) migrate ~6000km towards their spawning area in the Sargasso Sea. Based on the recent discovery that males swim even more efficiently than females, it was predicted that males also would be able to swim ~6000km within six months. Additionally, eels do not mature naturally in captivity due to strong neural inhibition. Earlier, it was hypothesized that swimming exercise is a natural trigger to induce sexual maturation and may even result in full maturation. In the present study two groups of farmed male silver eels were subjected to either endurance swimming or resting for up to 6months. It was found that male eels were able to swim continuously for a total distance of 6670km within 6months. The body weight decrease in swimming and resting males after 6months was similar (<30g) underlining the extreme low energy cost of swimming. In contrast to our expectation long-term swimming did not induce sexual maturation in farmed silver eels, suggesting that swimming alone is not sufficient as a trigger for sexual maturation. In conclusion, male eels are efficient long distance swimmers and likely able to cover the distance to the Sargasso Sea within the expected time span of 6months.

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 (n = 10) or swum (n = 10) 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.

Primitive duplicate Hox clusters in the European eel's genome.

The enigmatic life cycle and elongated body of the European eel (Anguilla anguilla L., 1758) have long motivated scientific enquiry. Recently, eel research has gained in urgency, as the population has dwindled to the point of critical endangerment. We have assembled a draft genome in order to facilitate advances in all provinces of eel biology. Here, we use the genome to investigate the eel's complement of the Hox developmental transcription factors. We show that unlike any other teleost fish, the eel retains fully populated, duplicate Hox clusters, which originated at the teleost-specific genome duplication. Using mRNA-sequencing and in situ hybridizations, we demonstrate that all copies are expressed in early embryos. Theories of vertebrate evolution predict that the retention of functional, duplicate Hox genes can give rise to additional developmental complexity, which is not immediately apparent in the adult. However, the key morphological innovation elsewhere in the eel's life history coincides with the evolutionary origin of its Hox repertoire.

Dramatic effect of pop-up satellite tags on eel swimming.

The journey of the European eel to the spawning area in the Sargasso Sea is still a mystery. Several trials have been carried out to follow migrating eels with pop-up satellite tags (PSATs), without much success. As eels are very efficient swimmers, tags likely interfere with their high swimming efficiency. Here we report a more than twofold increase in swimming cost caused by a regular small satellite tag. The impact was determined at a range of swimming speeds with and without tag in a 2-m swimming tunnel. These results help to explain why the previous use of PSATs to identify spawning sites in the Sargasso Sea was thus far unsuccessful.