Marine larval hatchery technology: Microbial management and immune system ontogeny in European eel

The European eel (Anguilla anguilla) was once a common fish in streams, lakes and fjords with considerable economic and social importance. It has a high market value and has been a dedicated target for both commercial fisheries and aquaculture in Europe. Since the turn of the century, however, fishing has been in decline, and the same applies to aquaculture, which is based on wild-caught glass eels. The glass eels, which are in a juvenile stage, are caught in targeted fisheries to supply farms with fry and assist in restocking. Today, the European eel population is under pressure due to historically low recruitment levels of glass eels and designated as "Critically Endangered" on the IUCN Red List. In response, ICES recommends a complete cessation of eel fishing at all stages to facilitate stock recovery. If we are to preserve the eel as a food fish, a sustainable solution would therefore be to develop hatchery techniques and close the eel's life cycle in captivity. Hatchery production of glass eels also has the potential to contribute to the management of European eel in the long term. However, establishing hatchery technology for breeding European eel in captivity is challenging due to the eel's complex life cycle and a distinct lack of knowledge about the reproductive stages and early life history in nature. The eel has life stages in the sea as well as in continental waters. The reproduction of European eel involves a migration over a long distance to the spawning area in the Sargasso Sea, where the earliest larval stages occur. From here, their larvae, the so-called leptocephalus larvae, are transported by ocean currents to the coasts of Europe and North Africa, where the leaf-shaped larvae transform into a juvenile stage, the glass eel. Glass eels, which are small and transparent, find their way into freshwater systems such as lakes, marshes and rivers or remain in coastal waters. Further on, they become yellow eels and later silver eels, which migrate to the Sargasso Sea to spawn. By the time they start the migration, eels are not sexually mature but held in a pre-pubescent stage through hormonal inhibition.

Due to the lack of insight regarding the eel's reproductive biology in nature and the needs of the early life stages, experimental research is the driving force in the effort to close the life cycle in captivity. Research over several decades for both the European and Japanese eel has created knowledge and progress in broodstock rearing, assisted reproduction, artificial fertilisation and egg quality, as well as the culture of the early life stages, which includes optimisation of culture systems, biophysical conditions and larval nutrition. For the European eel, this in the past decade led to a stable production of offspring that reach the feeding stage. Now, work on the establishment of larval culture, including the development of suitable larval feeds, is in focus but challenged by high mortality during early life stages. It is essential to solve this problem to close the life cycle and scale up production, but it remains unclear which factors or which combination of factors are responsible for the high mortality. In this context, the current PhD project focuses on the interaction between the bacteria in culture systems and offspring, concurrently investigating how the immune system of the offspring develops and functions under culture conditions. The project includes four separate studies based on controlled experiments, which together investigated how different treatments and culture conditions in captivity affected the composition of bacterial communities and the performance of offspring (e.g., survival, growth and development), while molecular studies of gene expression have provided new knowledge about the immune system development in the early stages of European eels.

In the first study, we investigated the importance of egg stocking density during incubation (varying from 500 eggs/L to 4000 eggs/L) for the survival of embryos and newly hatched larvae, and how it affected the bacteriome in the rearing water but also in the embryos and larvae, as well as their immune system responses. The study covered embryonic development from 0 hours post-fertilisation (hpf) until hatching (approx. 55 hpf), as well as the subsequent larval stages from hatching to 3 days post-hatch (dph). Hatching success was also recorded for the different incubation densities. The results showed higher survival at the lowest incubation density (500 eggs/L), but no effect on either hatching success or expression of genes related to immune or stress/repair responses. Mortality was highest during the embryonic incubation phase, while mortality decreased in newly hatched larvae. The high mortality identified at the highest incubation densities during the embryonic phase was associated with the presence of potentially detrimental bacterial taxa. Molecular studies of the expression of immune related genes indicated that the relatively undeveloped immune system in the eel's early life stages is probably unable to counteract the unfavourable microbial environment that occurs at high incubation densities. Although the lowest incubation density significantly improved offspring survival, it was a marginal improvement of 5% compared to the highest density. Coupled with the unaffected hatching success and molecular stress response at high incubation densities, it brings up practical considerations such as space requirements and workload in a hatchery. While mortality during the egg stage may be affected by several factors, the results indicate that effective management of bacterial communities during incubation and in the recirculating systems for larval culture is essential to prevent the selection for and proliferation of potentially harmful bacteria, especially in connection with larval feeding, which is initiated at the end of the yolk sac phase (approx. 10 dph).

It is particularly necessary to be able to control the r-selection in bacterial communities during larval cultures when initiating feeding. Research towards identifying the nutritional needs of European eel larvae struggles with a bottleneck in terms of larval survival especially from day 20 to 24, when high mortality indicates that the larvae have not ingested, digested and assimilated food in time. At the same time, the liquid diets used affect the bacterial communities. Therefore, the following study was conducted to investigate changes in water and larval bacteriomes as well as the larval immune response in a feeding experiment. The feeding trial included three different experimental diets (Diet 1, Diet 2 and Diet 3) offered to larvae aged 9 to 28 dph. Samples were collected for analysis of the bacteriome structure of water and larvae, as well as for analysis of the larvae's gene expression related to immune and stress response. Regardless of the diet, an increased expression of the gene hsp90 was found during the critical period from 20 to 24 dph, which indicates activated stress/repair mechanisms. At the same time, all diet groups showed a change towards a bacterial community in the larvae with reduced evenness (homogeneity) and increased occurrence of potentially harmful and opportunistic bacterial groups. This suggests that, in addition to food intake and nutrient quality, deleterious interactions between larvae and bacteria contribute to the marked mortality observed. Beyond the critical period, the highest survival was seen in larvae fed with Diet 3. In this group, on 22 dph, an upregulation of the genes tlr18 and c1qc, which code for actors in the recognition of pathogens and complement system, respectively, was registered. This upregulation suggests a molecularly more mature immune system with functional immuno-competence, probably enabling greater resistance to dominant harmful bacteria.

The third study was carried out as part of a subsequent trial, which aimed to improve the protocol for the first feeding of European eel larvae. Here, the focus was on optimising the amount of feed offered during larval culture, using the most promising diet (Diet 3), identified in the previous study. While large quantities of feed are assumed to promote food intake and growth in eel larvae, it may simultaneously promote the growth of fast-growing, opportunistic bacteria, thereby causing dysbiosis (imbalance) in the bacterial communities. In the experiment, we offered eel larvae a low (0.5 mL food/L water) or high (1.5 mL food/L water) amount of Diet 3. For both experimental groups, we investigated survival, food intake and growth of the eel larvae, but also the composition of larval and water bacteriomes, as well as the larvae's expression of genes related to food intake and immune and stress response between 9 and 30 dph. Despite a marginally lower survival at the beginning of the first-feeding period in the high-food group, survival was similar between the two groups at the end of the experiment. The group of larvae that received the high amount of food showed a higher degree of gut fullness, faster growth and a healthier bacteriome compared to the group that received the low amount. Analysis of bacterial communities indicated that bacteria associated with the diet affected the larval bacteriomes, especially in the group with high amount of food. Gene expression analysis showed that the gene encoding ghrelin, also called the hunger hormone, was upregulated on 30 dph in the low-food group, indicating that these larvae were starving compared to the high-food group. The expression profiles of immune and stress response genes showed no effect of the amount of food, indicating that feeding with high amounts of food should not negatively affect the larvae.

Research has previously pointed to a phase where the European eel larval immune system is molecularly still fairly undeveloped, which includes the period between hatching and ~8 dph when the larvae develop teeth. During this potentially immuno-compromised period, it is conceivable that they are vulnerable to interactions with harmful bacteria. Therefore, in the last study, β-glucan (BG), which is a known immunostimulant and modulator of bacterial communities, was tested for its prophylactic potential, especially during the yolk sac phase. Thus, yolk sac larvae were exposed to BG by adding a concentration of 5 mg/L to the water of the rearing tank. This was performed daily from 5 to 9 dph, after which the larvae entered the feeding stage and followed through the feeding period until 20 dph to assess any enduring effects. The larval survival, growth, development, expression of genes related to immunity and stress, and bacterial communities were tracked in comparison to a control group. The result showed no change in larval survival or growth, but for the BG-treated larvae a markedly lower rate of deformities was detected. Molecular analysis showed a downregulation of genes associated with anti-inflammatory response (il10) and stress (hsp90) in the BG-treated larvae. Furthermore, a positive alteration of the bacterial communities in both rearing water and larvae was seen in the BG-treated group. However, these positive effects were not long-lasting, as the difference evened out in 20-day-old larvae. The results indicate that BG treatment has the potential as an early prophylactic treatment in the culture of European eel larvae to reduce the incidence of deformities, modulate immune and stress response and positively alter the bacterial communities.

Altogether, this PhD project has generated new knowledge about bacterial community succession and changes in response to rearing practices and immune system functions as well as how these factors affect the offspring survival during the early life stages of cultured European eel. The eggs' maternal protective bacteriome appeared to be disrupted by common high density rearing practices in hatcheries, causing a dysbiosis that in turn affects the survival of offspring. The early life stages of eel do not have fully matured immune-related mechanisms to deal with microbial challenges, but we found that the immune system can be strengthened in later stages. Through this, resistance to harmful bacteria can potentially be improved. Here, for example, the inclusion of whey protein in larval diets and BG treatment can contribute to managing the bacterial communities and promoting the development of the immune system, which is crucial for improving the survival and quality of hatchery-produced eel larvae. The gained insight contributes to the improvement of offspring-rearing protocols but also emphasises the need for further research that can help reduce mortality during the embryonic stage and further elucidate the microbial dynamics in feeding larval culture to close the life cycle of this valuable fish species in captivity.