Effect of continuous dietary administration of the Saccharomyces cerevisiae yeast, Amnn9, on Pacific oyster (Crassostrea gigas) juveniles: Immunological and histopathological findings after Vibrio coralliilyticus challenge

Yeast cells and their cell wall components, mainly beta-glucans, are strong immunostimulants in bivalves, however, it remains unclear whether long-term administration of yeast contributes to prolonged activity of bivalve immune systems. Understanding these innate immune mechanisms is essential to establishing feeding strategies and disease prevention in hatcheries and nurseries. The present study examined the immunostimulatory effect of continuous dietary supply of the Saccharomyces cerevisiae mnn9 mutant (Amnn9), which exhibits elevated exposure of beta-glucans on the cell-wall surface, on Crassostrea gigas juveniles, and its protective effect against Vibrio coralliilyticus infections. In trial 1 the expression levels of immune-related genes were determined in oysters fed the Amnn9-containing diet 50A50Y (50% algae, 50% Amnn9; based on dry weight), compared to those fed the 100% algae-based diet (100A), after 6, 9, 12, 18, 24 h of feeding. In trial 2: the feeding period was expanded to 8 days, where the algal diet (100A) was provided every day, but substituted with 50A50Y for 0, 1, 2, 4, and 8 days (treatments 0D (control), 1D, 2D, 4D, and 8D, respectively) just before a Vibrio challenge, which was monitored for 7 days. Gene expression was measured before and 24-h after the Vibrio challenge. Histological analysis was performed on oysters on day 7 post-Vibrio challenge. Results showed that 50A50Y induced upregulation of interleukin 17-5 (CgIL17-5; 6 h), lysozyme (CgLys; 6 and 9 h), beta-glucan-binding protein (Cg beta GBP; 18 h), and superoxide dismutase (CgSOD; 18 and 24 h) genes in juveniles (trial 1). Before the challenge, trial 2 demonstrated overexpression of CgSOD in animals from 1D, CgLys in those from 1D, 2D, and 4D, but not in oysters from 8D, compared to those from 0D. However, higher CgIL17-5 expression levels were observed in animals from 2D, 4D, and 8D, suggesting that inflammatory genes can remain inducible after 8 days of Amnn9 feeding. After the Vibrio challenge, a significantly higher survival was observed in animals from 1D (35.8%), 2D (45.0%), 4D (49.2%), but not 8D (25.8%), compared to the survival of animals from 0D (16.7%). These findings suggest that regulatory feedback mechanisms occurring during continuous Amnn9 feeding might lead to host tolerance or homeostasis. Also, we showed that V. coralliilyticus promotes overexpression of Cg beta GBP, C-type lectin receptor (CgCLec-3), myeloid differentiation factor 88 (CgMyD88), nuclear factor-kappa B (CgNF kappa B), CgLys, and CgSOD genes in oysters at 24-h post-exposure. Finally, this study provides insights into histopathological manifestations in the digestive tract, muscles, and gills of Vibrio-infected oysters.