Recent insights into egg quality and larval vitality of the European eel Anguilla anguilla.

To date, the eel industry still depends on wild-caught juveniles that are grown to marketable size. There is an urgent need to close the eel life cycle in captivity to make aquaculture independent of the natural population. With this artificial reproduction protocol, yolk-sac larvae can be produced but egg quality may be impaired. Low survival rates and high deformity rates are frequently observed during the first week after hatching. Over the past four years, we have conducted studies with the aim to optimize the artificial reproduction protocol, thereby focussing on increasing egg and larval quality. Weekly carp or salmon pituitary extract (PE) treatment was successfully replaced with recombinant gonadotropins (rGTHs) to mature female eels and produce larvae. 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) was replaced with upstream precursor progesterone (P) to induce the endogenous production of DHP by the female eel. DHP and P were found equally potent in inducing oocyte maturation and ovulation. The effects of antibiotics on larval survival and the occurrence of deformities were investigated. Antibiotic treatment increased survival and decreased the occurrence of deformities indicating bacterial infection as an important cause. A deformity determination key for young eel larvae has been developed that provides a framework of reference for larval deformities which will be instrumental with gaining insights on the reasons behind each larval deformity. These improvements of the artificial reproduction protocol and hatchery practices will contribute to the production of robust eel larvae that survive, grow and metamorphose into juveniles that will later be able to reproduce in captivity.

The induction of oocyte maturation and ovulation in the European eel (Anguilla anguilla): in vitro and in vivo comparison of progesterone with 17α,20ß-dihydroxy-4-pregnen-3-one.

Ovulation in European eel is induced by injection of 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) as the maturation-inducing hormone (MIH). Female eels need to ovulate within 18 h after injection to release good quality eggs. Progesterone (P), as an upstream precursor of DHP, may promote endogenous DHP production and improve egg quality. The purpose of this study was therefore to compare treatment of P with DHP on batch level, in vitro, to determine dose-response effects, and in vivo, at a single dose. For the in vitro experiment, ovarian tissue was extracted and placed in culture plates containing hormone-free medium and media supplemented with the treatment: DHP at 1, 10 and 100 ng mL-1, or P at 10, 100 and 1,000 ng mL-1. At the start of incubation, the folliculated oocytes were sampled for histology, microscopy and qPCR. After incubation for 12 and 18 h, the oocytes were sampled for microscopy and qPCR analysis. For the in vivo experiment, females were either injected with DHP or P at a dose of 2 mg kg-1 to assess their effects on ovulation and reproductive success. At the moment of release, eggs were sampled for RNA sequencing to compare effects of DHP and P on the expression of genes involved in egg quality aspects. Remaining eggs were fertilized and larval viability was recorded. Both DHP and P were able to induce GVBD (DHP at 10 and 100 ng mL-1, P at 100 and 1,000 ng mL-1) in vitro. Expression of genes involved in oocyte maturation and ovulation was similar in vitro for both DHP and P treatments. Regarding the in vivo results, RNAseq results reflected similar DHP and P effects on the expression of genes involved in egg quality aspects. Females injected with either DHP or P ovulated, released eggs, and were equally able to produce larvae without any differences in reproductive success. Our results support the conclusion that DHP and P work equally well in vitro and in vivo. P is more attractive to apply as the price is 3,000 times lower than the price of DHP.

Steroid implants for the induction of vitellogenesis in feminized European silver eels (Anguilla anguilla L.).

Assisted propagation of the European eel will lead to a closed production cycle supplying the aquaculture industry with juvenile glass eels. Females require long-term weekly treatment with pituitary extract (PE), which is stressful and causes abnormalities in oogenesis. We tested the effects of 17α-methyltestosterone (17 MT), as potent androgen activating the androgen receptor, and 17ß-estradiol (E2), as an inducer of vitellogenesis, to shorten the duration of PE treatment.Four groups of feminized eels were subjected to a simulated migration and subsequent injection with implants containing 17 MT (17 MT-group), E2 (E2-group) or 17 MT plus E2 (17 MT + E2-group) to test for synergistic effects, or without any steroids as controls (C-group). The effects of a 2-months treatment were investigated by determining the eye index (EI), hepatosomatic and gonadosomatic index (HSI and GSI, respectively), plasma steroid concentrations by liquid chromatography mass spectrometry (LCMS), gonadal histology, expression of androgen receptors a and b (ara, arb); estrogen receptor 1 (esr1); FSH receptor (fshr); vitellogenin receptor (vtgr) and aromatase (cyp19), and the required number of weekly PE injections to fully mature. For many parameters, both the 17 MT and E2 groups showed an increase vs. controls, with the 17 MT + E2 group showing a synergistic effect, as seen for EI, GSI (3.4 for 17 MT and for E2, 6.6 for 17 MT + E2), oocyte diameter and ara, arb and esr1 expression. Concentrations of almost all focal steroids decreased with simulated migration and steroid treatment. Only eels of the 17 MT-group showed increased expression of cyp19 and of fshr, while fshr expression increased 44-fold in the 17 MT + E2 group, highlighting that co-implantation is most effective in raising fshr mRNA levels. Specific for eels of the E2 groups were vitellogenesis-associated changes such as an increase of HSI, plasma E2, and presence of yolk in the oocytes. Steroid treatments reduced the duration of PE treatment, again synergistically for co-implantation. In conclusion, E2 is necessary to start vitellogenesis, but 17 MT has specific effects on cyp19 and fshr expression. The combination is necessary for synergistic effects and as such, steroid implants could be applied in assisted reproduction protocols for European eel to improve oocyte quality leading to the production of more vital larvae.

What Goes Wrong during Early Development of Artificially Reproduced European Eel Anguilla anguilla? Clues from the Larval Transcriptome and Gene Expression Patterns.

In eels, large variations in larval mortality exist, which would impede the viable production of juvenile glass eels in captivity. The transcriptome of European eel larvae was investigated to identify physiological pathways and genes that show differential regulation between non-viable vs. viable larvae. Expression of genes involved in inflammation and host protection was higher, suggesting that non-viable larvae suffered from microbial infection. Expression of genes involved in osmoregulation was also higher, implying that non-viable larvae tried to maintain homeostasis by strong osmoregulatory adaptation. Expression of genes involved in myogenesis, neural, and sensory development was reduced in the non-viable larvae. Expression of the major histocompatibility complex class-I (mhc1) gene, M-protein (myom2), the dopamine 2B receptor (d2br), the melatonin receptor (mtr1), and heat-shock protein beta-1 (hspb1) showed strong differential regulation and was therefore studied in 1, 8, and 15 days post-hatch (dph) larvae by RT-PCR to comprehend the roles of these genes during ontogeny. Expression patterning of these genes indicated the start of active swimming (8 dph) and feed searching behavior (15 dph) and confirmed immunocompetence immediately after hatching. This study revealed useful insights for improving larval survival by microbial control and salinity reduction.

Author Correction: Rapid de novo assembly of the European eel genome from nanopore sequencing reads.

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Rapid de novo assembly of the European eel genome from nanopore sequencing reads.

We have sequenced the genome of the endangered European eel using the MinION by Oxford Nanopore, and assembled these data using a novel algorithm specifically designed for large eukaryotic genomes. For this 860 Mbp genome, the entire computational process takes two days on a single CPU. The resulting genome assembly significantly improves on a previous draft based on short reads only, both in terms of contiguity (N50 1.2 Mbp) and structural quality. This combination of affordable nanopore sequencing and light weight assembly promises to make high-quality genomic resources accessible for many non-model plants and animals.