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Yeast cells can play a crucial role in immune activation in fish and shellfish predominantly due to the cell wall component β-1,3-glucan, providing protection against bacterial or viral infections. However, the immunostimulatory capacity of dietary yeast cells remains poorly studied in bivalves. To understand the role of yeast cell wall components (mannan, β-glucan and chitin) as immune activators, this study characterized the surface carbohydrate exposure of the wild-type baker's yeast Saccharomyces cerevisiae (WT) and its Δmnn9 mutant, which presents a defective mannan structure, and compared these profiles with that of β-glucan particles, using fluorescein isothiocyanate (FITC)-labeled lectin binding analysis. Then, a first trial evaluated the immunological response in Crassostrea gigas juveniles after being fed for 24 h with an algae-based diet (100A) and its 50% substituted version (based on dry weight) with WT (50A50WT) and Δmnn9 (50A50Y), and the posterior resistance of the juveniles against Vibrio coralliilyticus infection (trial 1). The mRNA expression was measured for β-glucan-binding protein (CgβGBP), Toll-like receptor 4 (CgTLR4), C-type lectin receptor 3 (CgCLec-3), myeloid differentiation factor 88 (CgMyD88), nuclear factor-kappa B (CgNFκB), lysozyme (CgLys), interleukin 17-5 (CgIL17-5), and superoxide dismutase (CgSOD), in oysters, before and 24 h after the bacterial inoculation. A second trial tested the effect of incorporating Δmnn9 into the 100A diet for 24 h at different substitution levels: 0, 5, 10, 25, and 50% (100A, 95A5Y, 90A10Y, 75A25Y, and 50A50Y), followed by the bacterial challenge with V. coralliilyticus (trial 2). Our findings showed that the outer cell wall surface of WT is largely composed of mannan, while Δmnn9 presents high exposure of β-glucan and chitin, exhibiting similar FITC-lectin binding profiles (fluorescence intensity) to β-glucan particles. A significantly higher survival after the bacterial challenge was observed in oysters fed on 50A50Y compared to those fed 50A50WT and 100A in trial 1. This better performance of 50A50Y was supported by significantly higher gene expressions of CgLys, CgSOD, CgMyD88, and CgβGBP compared to 100A, and CgSOD and CgNFκB in relation to those fed on 50A50WT, prior to the bacterial inoculation. Furthermore, improved survival was observed in oysters fed 50A50Y compared to those offered lower Δmnn9 levels and 100A in trial 2. The superior performance of Δmnn9-fed oysters is mostly associated with the elevated presence of unmasked β-glucans on Δmnn9 cell wall surface, facilitating their interactions with oyster hemocytes. Further studies are needed to evaluate administration dose and frequency of Δmnn9 to develop strategies for long-term feeding., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

To predict the response of the European flat oyster ( Ostrea edulis ) and Pacific cupped oyster ( Crassostrea gigas/Magallana gigas ) populations to environmental changes, it is key to understand their life history traits. The Dynamic Energy Budget (DEB) theory is a mechanistic framework that enables the quantification of the bioenergetics of development, growth and reproduction from fertilization to death across different life stages. This study estimates the DEB parameters for the European flat oyster, based on a comprehensive dataset, while DEB parameters for the Pacific cupped oyster were extracted from the literature. The DEB parameters for both species were validated using growth rates from laboratory experiments at several constant temperatures and food levels as well as with collected aquaculture data from the Limfjorden, Denmark, and the German Bight. DEB parameters and the Arrhenius temperature parameters were compared to get insight in the life history traits of both species. It is expected that increasing water temperatures due to climate change will be beneficial for both species. Lower assimilation rates and high energy allocation to soma explain O. edulis' slow growth and low reproductive output. Crassostrea gigas' high assimilation rate, low investment in soma and extremely low reserve mobility explains the species' fast growth, high tolerance to starvation and high reproductive output. Hence, the reproductive strategies of both species are considerably different. Flat oysters are especially susceptible to unfavourable environmental conditions during the brooding period, while Pacific oysters' large investment in reproduction make it well adapted to highly diverse environments. Based on the life history traits, aquaculture and restoration of O. edulis should be executed in environments with suitable and stable conditions., (© The Author(s) 2022. Published by Oxford University Press and the Society for Experimental Biology.)

Green fluorescent protein (GFP) has been used extensively for in situ animal studies that follow up bacterial infection under epifluorescence microscopy. It is assumed that GFP is acting as a "neutral" protein with no influence on the bacterial physiology. To verify this hypothesis, the virulence of Vibrio splendidus ME9, Vibrio anguillarum NB10, and their respective GFP-tagged strains ME9-GFP and NB10-GFP (transconjugants) was compared in vitro and tested in vivo towards blue mussel (Mytilus edulis) larvae. Results showed that the incorporation of GFP negatively impacted the growth and swimming motility of NB10 in vitro. Correspondingly, the mRNA levels of genes involved in bacterial swimming motility (flaA, flaE, and cheR) were significantly down-regulated in NB10-GFP. As for the strain ME9 on the other hand, GFP incorporation only had a negative effect on swimming motility. However, both the strains NB10-GFP and ME9-GFP showed almost the same virulence as their respective parental strain towards mussel larvae in vivo. Overall, the data presented here demonstrated that incorporation of GFP may cause modifications in cell physiology and highlight the importance of preliminary physiological tests to minimize the negative influence of GFP tagging when it is used to monitor the target localization. The study also supports the idea that the virulence of Vibrio species is determined by complex regulatory networks. Notwithstanding the change of a single physiological trait, especially growth or swimming motility, the GFP-tagged Vibrio strain can thus still be considered usable in studies mainly focusing on the virulence of the strain. KEY POINTS: • The effect of GFP incorporation on physiological trait of Vibrio strains. • The virulence in vibrios could be multifactorial. • The stable virulence of Vibrio strains after GFP incorporation., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)