GAS1: A New ß-Glucan Immunostimulant Candidate to Increase Rainbow Trout (Oncorhynchus mykiss) Resistance to Bacterial Infections With Aeromonas salmonicida achromogenes.

ß-glucans are prebiotic and/or food additives used by the aquaculture industry to enhance the immune response of fish. Their efficiency may vary according to their origin and structure. In this study, the immunostimulant effects of two ß-glucan types extracted from wild-type baker's yeast (Saccharomyces cerevisiae) and its null-mutant Gas1 were investigated. Gas1 has a beta-1,3-glucanosyltransferase activity necessary for cell wall assembly. Using a positive (commercial product MacroGard®) and a negative control (a diet without glucans), we evaluated the immune responses and disease resistance of rainbow trout juveniles (mean weight, ~44 g) fed control, low (0.2%) and high (0.5%) doses of Macrogard®, Gas1, and Wild type-ß-glucan after a short-term (15 days, D15) or mid-term (36 days, D36) feeding periods. We found that ß-glucan supplemented diets did not affect growth performance, mortality, splenic index, or leukocyte respiratory burst activity on D15 nor D36. However, each ß-glucan triggered different immune effectors, depending of the doses or length of exposure compared to others and/or the negative control. Indeed, high dose of MacroGard® significantly increased lysozyme activities at D15 compared with the control and other diets (p<0.05). At D36, MacroGard ß-glucan enhanced the production of lymphocytes in comparison with the control diet (p<0.05). Regarding WT ß-glucan, at D36, WT-ß-glucan, especially the high dose, provided the highest enzymatic activities (lysozyme and ACH50) and Ig level (p<0.01). Furthermore, on D36, Gas1 also increased lysozyme activity, Ig proportion, and some immune genes (mcsfra, hepcidin) compared with MacroGard® (p<0.05). Besides, both doses of Gas1-ß-glucans increased the resistance of juveniles to bacterial infection highlighted by a higher survival rate at 14 days post-challenge compared with the control and other types and doses of ß-glucans (p<0.05). In conclusion, our results suggest that Gas1-ß-glucan could represent a promising immunostimulant that would help to prevent diseases in aquaculture even more efficiently than other ß-glucans already in use. Mode of action and particular efficiency of this new Gas1 mutant are debated.

Phloroglucinol Treatment Induces Transgenerational Epigenetic Inherited Resistance Against Vibrio Infections and Thermal Stress in a Brine Shrimp (Artemia franciscana) Model.

Emerging, infectious diseases in shrimp like acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus and mortality caused by other Vibrio species such as Vibrio harveyi are worldwide related to huge economic losses in industrial shrimp production. As a strategy to prevent disease outbreaks, a plant-based phenolic compound could be used as a biocontrol agent. Here, using the brine shrimp (Artemia franciscana) as a model system, we showed that phloroglucinol treatment of the parental animals at early life stages resulted in transgenerational inherited increased resistance in their progeny against biotic stress, i.e., bacteria (V. parahaemolyticus AHPND strain and V. harveyi) and abiotic stress, i.e., lethal heat shock. Increased resistance was recorded in three subsequent generations. Innate immune-related gene expression profiles and potential epigenetic mechanisms were studied to discover the underlying protective mechanisms. Our results showed that phloroglucinol treatment of the brine shrimp parents significantly (P < 0.05) enhanced the expression of a core set of innate immune genes (DSCAM, proPO, PXN, HSP90, HSP70, and LGBP) in subsequent generations. We also demonstrated that epigenetic mechanisms such as DNA methylation, m6A RNA methylation, and histone acetylation and methylation (active chromatin marker i.e., H3K4Me3, H3K4me1, H3K27me1, H3 hyperacetylation, H3K14ac and repression marker, i.e., H3K27me3, H4 hypoacetylation) might play a role in regulation of gene expression leading toward the observed transgenerational inheritance of the resistant brine shrimp progenies. To our knowledge, this is the first report on transgenerational inheritance of a compound-induced robust protected phenotype in brine shrimp, particularly protected against AHPND caused by V. parahaemolyticus and vibriosis caused by V. harveyi. Results showed that epigenetic reprogramming is likely to play a role in the underlying mechanism.

Effect of poly-ß-hydroxybutyrate on growth and disease resistance of Nile tilapia Oreochromis niloticus juveniles.

The growth promoting effect of the bacterial storage compound poly-ß-hydroxybutyrate (PHB) has been studied for young fish of high trophic level (European sea bass) and intermediate trophic level (Siberian sturgeon). Here, the effect of PHB on growth, digestive enzyme activities, body composition and diseases resistance of juvenile Nile tilapia (Oreochromis niloticus) of low trophic level was investigated. Although dietary PHB supplementation (5, 25 and 50 g PHB kg(-1) formulated semi-purified diet) during 28 days resulted in a trend of increased weight gain, there was no significant difference in the mean final body weight (258-284 mg) when compared to the fish from the control group (on average 218 mg). Lipase activity increased significantly with about 20-40% by the supplementation of PHB in the diet, which may have led to the significant increase in total lipid content with about 10% in the PHB treatment groups. However, the profile of total (n-6) fatty acids (FAs), total monounsaturated FAs and total saturated FAs relative to the total lipid was similar among various PHB treatments. An additional challenge test on gnotobiotic Nile tilapia larvae using the pathogen Edwardsiella ictaluri gly09R showed that feeding challenged larvae with PHB-enriched Artemia nauplii resulted in a 20% higher survival as compared to the challenged control larvae. Overall, it is suggested that the trend of increased body weight gain resulted from intestinal lipid digestion, absorption and deposition and that PHB is effective as an antimicrobial agent for application in Nile tilapia larviculture.

Immune response and disease resistance of shrimp fed biofloc grown on different carbon sources.

The objective of this study was to document the immunological effects of growing shrimp in biofloc systems. The experiment consisted of four types of biofloc systems in which bioflocs were produced by daily supplementation of four different carbon sources, i.e. molasses, tapioca, tapioca-by-product, and rice bran, at an estimated C/N ratio of 15 and a control system without any organic carbon addition. Each biofloc system was stocked with Pacific white shrimp (Litopenaeus vannamei) juveniles that were reared for 49 days. The use of tapioca-by-product resulted in a higher survival (93%) of the shrimp as compared to the other carbon sources and the control. The highest yield and protein assimilation was observed when tapioca was used as the carbon source. After 49 days, phenoloxidase (PO) activity of the shrimp grown in all biofloc systems was higher than that of the shrimp from the control system. Following a challenge test by injection with infectious myonecrosis virus (IMNV), the levels of PO and respiratory burst (RB) activity in the shrimp of all biofloc treatments were higher than that of the challenged shrimp from the control treatment. An increased immunity was also suggested by the survival of the challenged shrimp from the experimental biofloc groups that was significantly higher as compared to the challenged shrimp from the control treatment, regardless of the organic carbon source used to grow the bioflocs. Overall, this study demonstrated that the application of biofloc technology may contribute to the robustness of cultured shrimp by immunostimulation and that this effect is independent of the type of carbon source used to grow the flocs.

Reactive oxygen species generated by a heat shock protein (Hsp) inducing product contributes to Hsp70 production and Hsp70-mediated protective immunity in Artemia franciscana against pathogenic vibrios.

The cytoprotective role of heat shock protein (Hsp70) described in a variety of animal disease models, including vibriosis in farmed aquatic animals, suggests that new protective strategies relying upon the use of compounds that selectively turn on Hsp genes could be developed. The product Tex-OE® (hereafter referred to as Hspi), an extract from the skin of the prickly pear fruit, Opuntia ficus indica, was previously shown to trigger Hsp70 synthesis in a non-stressful situation in a variety of animals, including in a gnotobiotically (germ-free) cultured brine shrimp Artemia franciscana model system. This model system offers great potential for carrying out high-throughput, live-animal screens of compounds that have health benefit effects. By using this model system, we aimed to disclose the underlying cause behind the induction of Hsp70 by Hspi in the shrimp host, and to determine whether the product affects the shrimp in inducing resistance towards pathogenic vibrios. We provide unequivocal evidences indicating that during the pretreatment period with Hspi, there is an initial release of reactive oxygen species (hydrogen peroxide and/or superoxide anion), generated by the added product, in the rearing water and associated with the host. The reactive molecules generated are the triggering factors responsible for causing Hsp70 induction within Artemia. We have also shown that Hspi acts prophylactically at an optimum dose regimen to confer protection against pathogenic vibrios. This salutary effect was associated with upregulation of two important immune genes, prophenoloxidase and transglutaminase of the innate immune system. These findings suggest that inducers of stress protein (e.g. Hsp70) are potentially important modulator of immune responses and might be exploited to confer protection to cultured shrimp against Vibrio infection.

Effects of poly-ß-hydroxybutyrate (PHB) on Siberian sturgeon (Acipenser baerii) fingerlings performance and its gastrointestinal tract microbial community.

Poly-ß-hydroxybutyrate (PHB) is a natural polymer that can be depolymerized into water-soluble short-chain fatty acid monomers. These monomers can act as microbial control agents. In this study, the effects of partially replacing the diet of Siberian sturgeon fingerlings with 2% and 5% PHB were investigated. Replacing 2% of the diet with PHB improved weight gain, specific growth rate (SGR) and survival in the sturgeon fingerlings during the 10-week experimental period. Community-level physiological profiling and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) were used to analyze the microbial community diversity and community organization in the sturgeon gastrointestinal tract. DGGE analysis revealed that PHB affected the intestinal microbial species richness and diversity. The highest species richness was observed with 2% PHB. DNA sequencing of the dominant bands in 2% and 5% PHB treatments revealed that PHB stimulated bacteria belonging to the genera Bacillus and Ruminococcaceae. Principal component analysis, Lorenz curves and the Shannon index of Biolog Ecoplate data revealed that aerobic metabolic potential of the bacterial community was different in the PHB-treated fishes as compared with the control situation. Overall, our results indicate that PHB act as microbial control agents and replacement of 2% of Siberian sturgeon fingerling diet with PHB has beneficial effects.