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Knowledge on dietary nutrient requirements of first-feeding European eel larvae (Anguilla anguilla) is very limited. This study provides first ever information on in vivo lipid uptake and fatty acid (FA) metabolism of European pre-leptocephalus eel larvae and advances directions for dietary lipid and FA inclusions. The in vivo capability of eel larvae to incorporate and metabolize unsaturated fatty acids was tested on larvae at different ontogenetic stages (4, 8 and 12 days post hatch, DPH). Larvae were incubated in 10 mL flat-bottom tissue culture plates, with [1-14C]-labelled FA (18:2n-6, ALA; 18:3n-3, LA; 20:4n-6, ARA and 20:5n-3, EPA) directly added to seawater. The capability of the larvae for de-acylation and re-acylation of [1-14C]arachidonic acid (ARA), initially bound to phosphatidylcholine (PC) and phosphatidylethanolamine (PE), was also investigated. In all cases, control incubations without any radiolabelled substrate were performed for further lipid analysis. The results revealed that direct incubation with 14C-labelled FA is a feasible method to investigate in vivo FA and phospholipids metabolism of pre-leptocephalus stages of the European eel. No enzymatic elongation/desaturation activity towards [1-14C]C18 or [1-14C]C20 FA was detected. Consequently, ARA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) must be considered essential FA and thus provided firstly through female broodstock and later through diet at least during the first-feeding stage. Pre-leptocephalus larvae display a high capacity to remodel dietary phospholipids with a preferential esterification of all FA substrates into PC. The unexpectedly high esterification rate of [1-14C] ARA into PC and PE is supported by the individual FA profiles of the larval phospholipids. The high levels of ARA present in the European eel larvae denotes its physiological relevance for this species. It is therefore essential to consider this FA as particularly important when designing suitable broodstock – or first-feeding diets for this species.

Breeding European eel in captivity is a complex task. However, recent advances in assisted reproduction and culturing techniques now allow mass production of high-quality gametes, embryos, and first-feeding larvae. Here, three studies were conducted to determine whether dietary regime, chemoattractants, and light intensity have an impact on the incidence of larval first-feeding, gut fullness, and behavior. Firstly, larvae at 12, 16, and 20 days post-hatch (DPH) were allowed to forage on one of five diets, rotifer paste (RP), RP + cod roe, RP + octopus juice, RP + live rotifers, or Sargasso Sea plankton for 2 h and afterwards evaluated for presence or absence of ingested diet. Secondly, light effects on feeding at 15 and 16 DPH were tested, using the following intensities: High light at 21.5 ± 3.9 μmol m− 2 s− 1; intermediate at 6.8 ± 1.4 μmol m− 2 s− 1; low at 0.6 ± 0.2 μmol m− 2 s− 1; darkness; and a control where no feed was added in darkness. Lastly, behavioral observations were recorded on larvae at first-feeding. Results showed no evidence of first-feeding at 12 DPH. At 16 DPH, 23–50% of larvae had ingested feed when offered either RP, RP + live rotifers, or RP + cod roe. At 20 DPH, the highest incidence of feeding was detected when larvae were fed RP and chemoattractants; 8–30% of larvae ingested feed. Light had a positive impact on feeding at 15 and 16 DPH, such that incidence of first-feeding and gut fullness increased at the intermediate to high light intensities, where up to 50% of larvae ingested the RP diet and had 9% gut fullness. Swimming of larvae was characterized by bouts produced by undulations of the caudal region, followed by pauses where larvae remained motionless. Duration of swimming increased from 41 to 218 s at 13–17 DPH, respectively. Larvae exposed to RP-based diets swam more than the control (no feed). Larval attacks were observed in benthic and pelagic zones of the tank, however were more frequently observed in the benthos. Here, 8–10%, 60–73% and 69–86% of larvae were observed grazing on RP-based diets at 13, 15 and 17 DPH, respectively. Typically, attacks were associated with S-bend posture and upon initiation of S-bends the larvae remained stationary before an explosive lunge. At 13–17 DPH, larvae fed the RP-based diets exhibited a higher incidence of attacks than the control. Together, this work provides benchmark diets and conditions for future feeding and growth trials. Statement of relevance First-feeding by European eel larvae in captivity.

Captive breeding of European eel, Anguilla anguilla is challenged by the complex hormonal control of Anguillid eel reproduction and the distinctive ontogeny of the leptocephalus larvae that are unique to the Elopomorph superorder. Recent experimental research has succeeded in the production of viable eggs and larvae of European eel, providing the basis for studies on early life stages of this species in captivity. In this study, we describe and illustrate morphological characteristics of eggs, embryos, and larvae from fertilization to termination of the yolk sac stage and provide a comparison with additional commercially important eel species. Furthermore, we model growth during the critical first phase in larval ontogeny, i.e. the yolk sac stage, and test for maternal effects. The eggs of A. anguilla typically have numerous oil droplets that coalesce into a single large oil droplet, while the zygote forms a large perivitelline space, reaching an egg diameter of 1.45 ± 0.12 mm at 3.0 to 3.5 h post fertilization. Embryonic development from fertilization to larval hatch lasted ~ 46–48 h at 20 °C with the larvae emerging in a relatively undeveloped stage with a protuberant yolk sac. During the period of yolk and oil absorption, the larvae undertook significant changes in head and body morphology. At the completion of yolk sac absorption, the largely transparent larvae had a set of protruding teeth, pigmented eyes and tail, and a simple alimentary tract. Larvae appeared capable of feeding at ~ 12 days post hatch at 20 °C, and were able to survive another ~ 10 days without feeding. Larval length approached an asymptotic maximum of 6.8 mm around day 10 in non-fed larvae. Larval batches from different maternal origins varied in yolk sac size and the extent of yolk sac resources influenced larval size at the end of the yolk sac stage. The ontogenetic description presented here fills a gap in knowledge about the yet undiscovered early life stages of native European eel, which can provide a framework of reference for the development of hatchery technology. Such progress is urgently needed for a self-sustained aquaculture of this high-value and critically endangered species. Statement of relevance European eel is a high-value species in aquaculture, however, production is presently hampered by reliance on wild caught fry. Captive production of glass eels will reopen markets in Europe and Asia, benefiting European eel producers. The results presented here document recent progress within assisted reproduction and larval culture of this species in aquaculture and aid establishing hatchery technology of this species.

Production of high-quality offspring from farm-raised broodstock is fundamental to establish a closed-cycle hatchery production of European eel, Anguilla anguilla. While development of larval culture technologies progresses, the present study focused on effects of essential fatty acid (EFA) composition of eggs on offspring quality. Three reproduction experiments were conducted, two of which included farm-raised broodstock fed different diets for different periods of time and one wild-caught broodstock, using size-matched females. The formulated diets varied in levels and ratios of three essential fatty acids, arachidonic acid (20:4n-6; ARA), eicosapentaenoic acid (20:5n-3; EPA), and docosahexaenoic acid (22:6n-3; DHA), while feeding periods lasted either 55 or 79 weeks. Dietary influences on egg and offspring fatty acid composition and offspring quality were evaluated and results of the most successful dietary regime was compared to those of wild-caught females. Results showed that elevated dietary levels of ARA were reflected in unfertilized eggs, with high ARA diets significantly increasing the amounts of floating eggs, total lipid content in eggs, fertilization success, and embryonic survival. Further EFA enhancements and prolonged feeding resulted in higher ARA and lower EPA levels in the unfertilized eggs, while DHA levels did not change. Females with prolonged feeding produced offspring of higher quality, i.e. higher egg dry weight and larval survival. Overall, offspring of farm-raised females showed higher EFA levels than those of wild-caught females. However, while fertilization success was comparable, offspring of farm-raised females had significantly lower embryonic survival and hatch success as well as higher proportions of cleavage abnormalities. These results identified embryonic development as the main bottleneck in offspring production from farm-raised females. Once hatched, larval survival and quality was comparable between farm-raised and wild-caught females. Notably, enhancement of essential fatty acids in female broodstock diets in combination with a long feeding period improved the production of high quality offspring.

A presumed parasitic protozoan was found in the eggs of European eel obtained from an experiment on captive breeding of eel, Anguilla anguilla, based on silver eels from a freshwater lake in the northern part of Denmark. Gross morphology of the organism was comparable to that of early stages of Ichthyodinium, a syndinian dinoflagellate parasite found in pelagic eggs of various marine fish species. Sequences of genes coding for small subunit ribosomal RNA confirmed that the organism was an Ichthyodinium species, and molecular phylogenetic analysis demonstrated the presence of two Ichthyodinium genotypes: one occurring in the Atlantic Ocean and adjacent coastal waters and one in the Pacific Ocean area. The inclusion of several GenBank-derived environmental gene sequences, from the Caribbean Sea, revealed to represent Ichthyodinium, suggesting that this parasite genus is ubiquitous in the World's oceans.

Recent experimental research applying hormonally induced maturation in European eel has resulted in production of viable eggs and yolk-sac larvae. However, present incubation and larval rearing conditions are suboptimal and few larvae survive until onset of first feeding. The aim of this work was to investigate if high mortality during egg incubation and larval culture resulted from microbial interference. By suppressing microbial coverage and activity on fertilised eel eggs using antibiotic and disinfection treatment, egg hatching success and larval longevity were significantly improved. A new approach based on scanning electron microscopy was developed to quantify microbial coverage of eggs. Measurements of microbial coverage in combination with growth curves of egg-associated bacteria indicated that microbial activity rather than physical coverage led to reduced hatch success. In addition, an inverse relationship between microbial coverage of eggs and larval survival indicated that attachment of micro-organisms on the egg surface during the last 24 h of incubation affected later larval survival. These results suggest that microbial control through application of egg surface disinfection in combination with microbial management will be fundamental for improved post-hatch larval survival.