Introduction and Aim
European seabass (Dicentrarchus labrax), an important mediterranean aquaculture species, is increasingly threatened by bacterial pathogens, including the gram-negative Photobacterium damselae subsp. piscicida. Photobacteriosis outbreaks can decimate entire productions, leading to massive losses and economic costs. Limited strategies can be employed on the mitigation of such outbreaks. A promising approach for the industry are functional feeds as prophylactic alternatives. Functional diets with algae derivatives can enhance the immune status and growth, helping in the prevention and outbreak mitigation of pathogen-related diseases.
This study aimed to evaluate how aqueous extracts of Tetraselmis chui and Ulva sp. combined with established commercial immunostimulants, a β-glucan-rich product and a sulphated polysaccharide product, modulate the immune response and disease resistance after a short term feeding boost followed by a pathogen insult.
Diet composition and experimental design
This study is comprised of two stages: 1) feeding trial, where fish were fed 4 experimental diets for 5 days. Fish were fed a control diet (CTRL) based on commercial formulations, the CTRL supplemented with a combination of an aqueous extract of Tetraselmis chui and, either, the commercially available supplement rich in β-glucans (Diet 1), or sulfated polysaccharides (Diet 2), or CTRL supplemented with a blend of Ulva sp. extract and the β-glucans rich product (Diet 3). Inclusion levels were within the range practiced by the industry for feed additives (ppt to ppm). Seabass were fed by hand at 3% fish body weight per day; 2) After the feeding trial, fish were orally infected with a pathogenic strain of Photobacterium damselase subsp. piscicida (strain: GRDL 19-1 (HK2)), and mortalities were monitored for 8 days. Fish were sampled at three sampling points, before infection, to assess the basal immune status after being fed dietary treatments, and at 6- and 72 hours after pathogen insult, to evaluate the immune respose to infection. Liver and head-kidney samples were collected for gene expression analyses, anterior intestine for proteomics, liver for oxidative status, and plasma for humoral immune parameters.
Results and Discussion
Proteomic analysis of the anterior gut showed major modulation 72 hours after pathogen exposure, with major changes in proteins associated with gut integrity and metabolic pathways (figure 1). Gene expression of immune genes in the head-kidney showed a significant modulation with time after pathogen challenge, particularly at 72 hours after infection. Gene expression analysis of liver showed a dietary modulation in genes associated with fatty acid metabolism and energy production at the end of the feeding trial. It was observed a significant upregulation of these pathways-related genes in fish fed the experimental diets when compared to CTRL, being considerably higher in fish fed Diet 2. Genes related to pathogen recognition were also upregulated in the liver after feeding Diet 2. Taken together, data from the present studysuggest that supplementing Tetraselmis chui extracts with sulfated polysaccharides can have a synergistic effect in enhancing fish basal immune status. However, in the presence of Photobacterium damselase subsp. piscicida, dietary modulation was not sufficient to elicit a differential response, as it was observed in the proteomic analysis and visual observation where it was noted a intestinal tissue degradation at 72 hours. These findings are consistent with disease resistance data observed after challenge, where fish survival ranged from 30.8% in Diet 3 to 17% in Diet 2 after 8 days, with no significant differences observed between treatments.
Figure 1 - Bubble chart of top 10 KEGG enrichemnt analysis (per diet) of differential abundant proteins (DAP) in the anterior gut of European seabass-fed experimental diet (Control, Diet 1, Diet 2 and Diet 3) 72 hours after pathogen exposure.
Acknowledgments
This work was financially supported by "Pacto da Bioeconomia azul" (Project No. C644915664-00000026) within the WP5 Algae Vertical, funded by Next Generation EU European Fund and the Portuguese Recovery and Resilience Plan (PRR), under the scope of the incentive line "Agendas for Business Innovation" through the funding scheme C5 - Capitalization and Business Innovation. Andr�� Cunha was supported by Funda����o para a Ci��ncia e Tecnologia (2022.12761.BD).