Effects of European eel egg and larval stocking density on rearing water, offspring bacteriome and derived immune response

If human exploitation of the “critically endangered” European eel (Anguilla anguilla) is to continue, the eel's life cycle in captivity must be closed and techniques established to produce viable offspring in hatcheries. Despite stable offspring production, high mortality rates during early developmental stages have hindered progress in closing the lifecycle in captivity. We hypothesised that mortalities affecting embryos and newly hatched larvae, when the immune system is expected to play a vital role in protecting the offspring, could be linked to adverse microbial conditions arising in high-density rearing. We therefore set out to explore the bacteriome changes in rearing water and offspring as well as the molecular immunologic development of offspring at different initial stocking densities (500, 1000, 2000, and 4000 eggs/L) from fertilisation to 3 days post-hatch (dph). The lowest density resulted in ~5% higher survival. Independent of density, most mortality (~75%) occurred during embryonic rather than larval development. The bacteriomes of both offspring and rearing water differed according to stocking density, with a potentially healthier bacteriome at the lowest density, marked by greater ASV richness and abundance from various phyla (Proteobacteria, Actinobacteria, Fusobacteria, and Firmicutes). In contrast, the bacteriome of the highest density was dominated by Proteobacteria ASVs, including the potentially harmful Vibrionales order. We observed stage-specific bacteriome changes in offspring, transitioning from an inherent and balanced bacteriome in unadulterated eggs to a dominance of rapid-growing opportunistic bacteria in embryos (48 hpf), and finally to a more diverse bacterial community in larvae (3 dph). Thus, host-microbe interactions appear to significantly impact the overall high mortality observed during embryonic development. As the expression of stress and immune related genes was not affected by density, it seems that the molecular immune system was incapable of handling microbial interference during these early stages, aggravating the challenges faced during hatchery production. We conclude that low-density (500 eggs/L) incubation is a valid strategy to improve survival. However, high stocking densities (up to 4000 eggs/L) might be an acceptable compromise allowing more efficient utilisation of infrastructure and labour. At 4000 eggs/L, survival was reduced by only ~5%, while there was no effect on hatching success and no cellular stress response. Nevertheless, bacterial community management tools should be in place during high-density incubation to avoid r-selection and thus counter bacteria-associated mortality.