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.

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.

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.

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

BACKGROUND: Studies 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. RESULTS: A 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. CONCLUSIONS: In 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.

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 (n = 10) were able to swim at a critical swimming speed (U(crit)) of 0.548±0.007 m s(-1) or 18.0 standard body lengths (BL) s(-1). The optimal swimming speed (U(opt)) 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 U(crit). The cost of transport at optimal swimming speed (COT(opt)) was 25.23±4.03 µmol g(-1) m(-1). A group-wise experiment was conducted with zebrafish (n = 83) swimming at U(opt) for 6 h day(-1) for 5 days week(-1) for 4 weeks vs. zebrafish (n = 84) 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.

Zebrafish development and regeneration: new tools for biomedical research.

Basic research in pattern formation is concerned with the generation of phenotypes and tissues. It can therefore lead to new tools for medical research. These include phenotypic screening assays, applications in tissue engineering, as well as general advances in biomedical knowledge. Our aim here is to discuss this emerging field with special reference to tools based on zebrafish developmental biology. We describe phenotypic screening assays being developed in our own and other labs. Our assays involve: (i) systemic or local administration of a test compound or drug to zebrafish in vivo; (ii) the subsequent detection or "readout" of a defined phenotypic change. A positive readout may result from binding of the test compound to a molecular target involved in a developmental pathway. We present preliminary data on assays for compounds that modulate skeletal patterning, bone turnover, immune responses, inflammation and early-life stress. The assays use live zebrafish embryos and larvae as well as adult fish undergoing caudal fin regeneration. We describe proof-of-concept studies on the localised targeting of compounds into regeneration blastemas using microcarriers. Zebrafish are cheaper to maintain than rodents, produce large numbers of transparent eggs, and some zebrafish assays could be scaled-up into medium and high throughput screens. However, advances in automation and imaging are required. Zebrafish cannot replace mammalian models in the drug development pipeline. Nevertheless, they can provide a cost-effective bridge between cell-based assays and mammalian whole-organism models.