Introduction
Tryptophan has been shown to play several roles in modulating stress and immune responses in fish, and it is hypothesized that may also serve as a complementary strategy to vaccination in aquaculture. Th is study evaluates the combined effects of dietary tryptophan supplementation and dip vaccination on skin and intestinal mucosal immunity of European seabass ( Dicentrarchus labrax), as well as the complex interaction between neuroendocrine-immune systems.
Materials and Methods
European seabass juveniles (26.23 ± 7.22 g) were randomly distributed into two different recirculating seawater systems (temperature 20 ± 0.5 °C) . In a complete randomized design, fish were fed either a control diet (CTRL) or one of two CTRL-based diets supplemented with tryptophan (TRP1 0.05 % DM and TRP2 0.5 % DM) in triplicate tanks per system. After 3 days of feeding, 3 fish per treatment from system 1 were euthanized by an overdose of 2-phenoxyethanol and skin mucus and blood (immune status) , brain (diced in hypothalamus), pituitary gland, head-kidney (gene expression) and intestine ( shotgun proteomics) samples were collected . The remaining fish were dip vaccinated, returning to their original tanks, and sampled after 1 hour, 6 hours, and 21 days post-vaccination. Following vaccination, fish were fed the same diets for 3 more days and after that, all fish were fed the CTRL diet until the end of the trial (21 days). Fish from system 2 were undisturbed following the same dietary regime of system 1.
Results and Discussion
Upon vaccination procedures, an expected inflammatory response was observed in fish fed CTRL and TRP2 diets 1 and 6 hours after , as shown by higher plasma cortisol levels , reduced total immunoglobulin M levels , and increased expression of neuroendocrine markers (htr2a and tph1α) . In contrast, this inflammatory response was attenuated in dip-vaccinated fish fed TRP1, as evidenced by low transcription rates of serotonin-related genes , reduced plasma cortisol, and enhanced bactericidal activity in skin mucus - overall displaying a physiological profile similar to undisturbed control fish. Notably , dip- vaccinated fish fed TRP1 and sampled 6 hours post-vaccination exhibited a gut proteomic profile resembling also those of the undisturbed CTRL group. These findings suggest that tryptophan at low supplementation levels may mitigate the stress-related effects of dip vaccination and potentially enhance vaccine efficacy.
Regarding tryptophan supplementation’s longstanding effects on fish vaccination efficacy, the lowest supplementation dose counteracted vaccine-mediated reduced gene expression (c3 , ido2 , igm and il1β ), again mirroring the gene expression patterns observed in undisturbed fish. This suggests a sustained beneficial effect of tryptophan, even after a short administration period, with effects persisting up to 15 days after the end of the treatment.
Conclusions
Overall, the findings highlight a complex interplay between tryptophan supplementation and immune responses , supporting its potential as an effective dietary strategy to enhance vaccination outcomes in aquaculture.
Keywords: Fish physiology, inflammatory response, nutritional immunology, tryptophan, vaccination
Acknowledgements
This work was supported by IMMUNAA project (PTDC/CVT-CVT/7741/2020), financed through Fundação para a Ciência e a Tecnologia (FCT). DP, IC, BC, and RA were supported by FCT, Portugal (UI/BD/150900/2021, 2021.04867.BD, 2020.00290.CEECIND and 2022.03248.CEECIND, respectively).