European eel larval quality and feeding regime: Establishing first-feeding culture

Development of closed-cycle hatchery technology is pivotal for sustainable aquaculture production of the European eel (Anguilla anguilla). In this context, the research conducted within this PhD project contributes to generating the knowledge needed to establish larval culture of European eel by addressing biochemical, nutritional and developmental factors influencing early larval stages. This involves experimental studies, which use an integrative set of tools to elucidate relations between offspring survival, developmental indicators and molecular mechanisms in the pursuit of insights into the early life history of the enigmatic eel larvae. The thesis frames the outcomes of four studies within three main topics: i) larval quality and biochemical composition, ii) maturation of the gut during the transition between endogenous and exogenous feeding and iii) first-feeding diets and dietary regimes for eel larval culture. All those aspects influence larval development and survival thereby being vital for advancing the establishment of feeding larval culture of European eel.

In aquaculture, variable egg quality from captive broodstock is a common problem affecting viable offspring production. A stable supply of high quality eggs requires comprehensive understanding of the factors that regulate gamete and offspring quality. For the European eel, reproductive protocols involve administration of pituitary extracts from carp (CPE) or salmon (SPE) to induce and sustain oocyte development and vitellogenesis. During vitellogenesis that lasts several months, yolk constituents (for example protein, lipid, and amino acid) are incorporated into the oocytes. In this regard, Study 1 compared the effects of CPE and SPE on the biochemical composition of eggs and yolk sac larvae as well as the subsequent nutrient utilization by the developing larvae during the yolk-sac stage. The results showed that the type of pituitary extract used to induce vitellogenesis in female eel influenced the biochemical composition of the offspring, where SPE females produced more buoyant eggs with higher fertilization rate as well as larger larvae with more energy reserves, while CPE females spawned eggs with higher lipid and free amino acid content. Whereas these differences were marked at the egg, embryo and early larval stages, the composition of surviving larvae originating from the two treatments were similar by the end of the yolk sac stage. Overall, the yolk sac larvae utilized lipids and monounsaturated fatty acids as energy source, until they reached 8 days post hatch (dph), marking the time point, where larvae would benefit from the presence of exogenous nutritional stimulants, possibly such as gut-priming agents or prefeeding protocols, in order to meet subsequent energy and constituent requirements.

European eel larvae reach the first feeding stage around 10-12 dph at 18-20°C, which is characterized by attainment of a fully functional feeding apparatus as well as vision. However, molecular processes related to digestion and food intake are starting to be expressed already at 4 dph. At this point, introduction of gut priming agents or prefeeding may advance the transition from endogenous to exogenous feeding by stimulating digestive functions. Accordingly, in Study 2, commercially available gut priming agents were tested, including prebiotics, probiotics and their combination, i.e. synbiotics, administered to the larvae during the endogenous feeding stage. Results revealed an increased mortality, when eel larvae were offered synbiotics, while both probiotics and synbiotics impaired somatic growth. Nevertheless, gene expression analyses indicated a prospective adaptive capacity towards earlier maturation of the larval gut, enhancing digestive capacity. The gut-priming agents and regimes applied in this experiment did not appear suited for eel larvae in culture, however, also water management strategies, when adding nutrients to larval culture, need attention e.g. avoiding overload of the recirculating system or the formation of debris on the tanks bottom, as microbial growth appears to interfere with larval survival.

In parallel, Study 3 investigated the effect of early introduction of feed (prefeeding) on the transition from the yolk sac stage to active feeding larvae. Since a suited first feeding diet has not yet been developed for Euroepean eel larvae, three different feeds and feeding regimes were tested during the yolk sac stage and throughout the first feeding window. The results indicated that while prefeeding challenged larval survival, the early introduction of nutrients might benefit the functionality of the gastro-intestinal tract and equip the larvae with an improved digestive capacity. Although, none of the feeding regimes tested seemed to include a sufficiently balanced diet resulting in larval growth, one of the feeding regimes applied in this study resulted in the highest survival rates registered at that time point. In particular, the molecular results added insights of importance to the development of diets for first feeding culture of European eel larvae, as shown in one of the Feeding regimes, where gene expression together with survival data pointed towards a dietary composition, getting closer to the nutritional requirements of first-feeding European eel larvae.

In Study 4, three formulated diets were tested experimentally, providing the feeds to larval cultures from 10 dph, i.e. the onset of the first feeding window until 28 dph. The three diets, taking inspiration from larval feeds applied for Japanese eel (A. japonica), shared the same basic ingredients. Here, Diet 1 represented the simplest composition, while Diet 2 and 3 were modifications, where proteins with different molecular size were added. Thus, Diet 2 included fish hydrolysate with ~3 kDa protein size, while Diet 3 included whey protein of ~10 kDa. The results showed an early benefit (15 dph) of Diet 1 in terms of initially improved larval survival, morphological development and gene expression. However, at 22 dph, larvae fed Diet 3 showed upregulated expression patterns of the genes encoding the major digestive enzymes, a downregulation of ghrelin, encoding the “hunger hormone”, together with an improved survival and growth. Moreover, the expression of digestion, food intake and growth related genes continued to increase towards 28 dph for larvae fed Diet 3. Thus, it seems that the inclusion of more complex dietary proteins (as in Diet 3), but not hydrolyzed peptides (as in Diet 2) is beneficial in order to promote larval ontogenetic performances after the successful transition to exogenous feeding. In spite that survival was limited, larvae fed Diet 3 survived throughout and for the first time, beyond the first feeding stage, providing important information in relation to the nutritional requirements of first feeding larvae of this species. The results, therefore, provide a useful basis for future research efforts towards establishment of larval culture of European eel.

Altogether, the combination of methods applied in this PhD project has provided novel insights into the biochemistry, resource utilization, developmental physiology and nutritional requirements of European eel larvae in culture. Still, the establishment of a first feeding culture of European eel is at a pioneering state. In the present studies, based on consecutive targeted experiments, larval survival and growth has been incrementally improved and insights substantiated. It is the first time that successfully feeding leptocephalus larvae survived beyond the first feeding stage enabling a morphological and molecular characterization of development. These results pave the way for forthcoming extended feeding experiments together with integrating multi- and interdisciplinary research tools that will benefit hatchery development and conservation of the critically endangered European eel.