Due to the negative consequences associated with the use of antibiotics, researchers, and food producers have studied alternatives, such as probiotics, for the control of fish diseases. The probiotic properties of yeasts in aquaculture have been scarcely considered. The present study investigated the probiotic properties of local yeast strains for aquaculture application in the protection of bacterial diseases. Yeast strains (n = 15), previously isolated from the intestinal gut of healthy salmonids, yellowtail, and croaker, were evaluated for their protection of zebrafish larvae following a Vibrio anguillarum challenge. We developed an infection model on zebrafish larvae with V. anguillarum, observing rapid mortality (≥50%) 5 days post-immersion challenge. Infection of Tg(Lyz:DsRed)nz50 larvae with fluorescent-marked V. anguillarum showed the oro-intestinal as the natural route of infection concomitant with an inflammatory response of the larvae reflected by neutrophil migration outside the hematopoietic tissue. Thirteen of 15 strains increased the percentage of larvae survival after the V. anguillarum challenge, although no yeast showed in vitro anti-V. anguillarum activity. In a subset of yeasts, we explored yeast–larvae interactions using fluorescent yeast and evaluated larvae colonization by culture analysis. All fluorescent yeasts were located in the gastrointestinal tract until 5 days post-inoculation (dpi). Yeasts reached 103 CFU/larvae at 0 dpi, although the persistence until 5 dpi of the viable yeast in the gut was different among the strains. These results reveal that some yeasts isolated from the gut of fish could be potential probiotics, reducing the mortality associated to V. anguillarum challenge, and suggest that gut colonization could be involved in the protective effect. Future studies should elucidate other mechanisms involved in yeast protection and verify the beneficial effects of probiotic use in commercial fish species.

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We investigated mechanisms involved in the protection of zebrafish (Danio rerio) larvae by two probiotic candidate yeasts, Debaryomyces hansenii 97 (Dh97) and Yarrowia lypolitica 242 (Yl242), against a Vibrio anguillarum challenge. We determined the effect of different yeast concentrations (104–107 CFU/mL) to: (i) protect larvae from the challenge, (ii) reduce the in vivo pathogen concentration and (iii) modulate the innate immune response of the host. To evaluate the role of zebrafish microbiota in protection, the experiments were performed in conventionally raised and germ-free larvae. In vitro co-aggregation assays were performed to determine a direct yeast-pathogen interaction. Results showed that both yeasts significantly increased the survival rate of conventionally raised larvae challenged with V. anguillarum. The concentration of yeasts in larvae tended to increase with yeast inoculum, which was more pronounced for Dh97. Better protection was observed with Dh97 at a concentration of 106 CFU/mL compared to 104 CFU/mL. In germ-free conditions V. anguillarum reached higher concentrations in larvae and provoked significantly more mortality than in conventional conditions, revealing the protective role of the host microbiota. Interestingly, yeasts were equally (Dh97) or more effective (Yl242) in protecting germ-free than conventionally-raised larvae, showing that protection can be exerted only by yeasts and is not necessarily related to modulation of the host microbiota. Although none of the yeasts co-aggregated with V. anguillarum, they were able to reduce its proliferation in conventionally raised larvae, reduce initial pathogen concentration in germ-free larvae and prevent the upregulation of key components of the inflammatory/anti-inflammatory response (il1b, tnfa, c3, mpx, and il10, respectively). These results show that protection by yeasts of zebrafish larvae challenged with V. anguillarum relates to an in vivo anti-pathogen effect, the modulation of the innate immune system, and suggests that yeasts avoid the host-pathogen interaction through mechanisms independent of co-aggregation. This study shows, for the first time, the protective role of zebrafish microbiota against V. anguillarum infection, and reveals mechanisms involved in protection by two non-Saccharomyces yeasts against this pathogen.

Intestinal inflammation is a harmful condition in fish that can be triggered by the ingestion of soybean meal. Due to the positive costs-benefits ratio of including soybean meal in farmed fish diets, identifying additives with intestinal anti-inflammatory effects could contribute to solving the issues caused by this plant protein. This study evaluated the effect of incorporating lactoferrin (LF) into a soybean meal-based diet on intestinal inflammation in zebrafish. Larvae were fed with diets containing 50% soybean meal (50SBM) or 50SBM supplemented with LF to 0.5, 1, 1.5 g/kg (50SBM+LF0.5; 50SBM+LF1.0; 50SBM+LF1.5). The 50SBM+LF1.5 diet was the most efficient and larvae had a reduced number of neutrophils in the intestine compared with 50SBM larvae and an indistinguishable number compared with control larvae. Likewise, the transcription of genes involved in neutrophil migration and intestinal mucosal barrier functions (mmp9, muc2.2, and β-def-1) were increased in 50SBM larvae but were normally expressed in 50SBM+LF1.5 larvae. To determine the influence of intestinal inflammation on the general immune response, larvae were challenged with Edwardsiella tarda. Larvae with intestinal inflammation had increased mortality rate compared to control larvae. Importantly, 50SBM+LF1.5 larvae had a mortality rate lower than control larvae. These results demonstrate that LF displays a dual effect in zebrafish, acting as an intestinal anti-inflammatory agent and improving performance against bacterial infection.

Because of its outstanding biological and industrial importance, many efforts have been made to characterize the mycobiota of new environments and their biochemical and biotechnological potentials. Gut mycobiota can be a source of novel yeasts with the potential to be used as probiotics or have industrial applications. In this work, we characterized two as-yet unexplored yeast communities from the intestinal content of the cultured marine Chilean fishes Genypterus chilensis (G. chilensis) and Seriolella violacea (S. violacea). Yeasts were isolated through culture, identified by sequencing their ITS region, and characterized their enzymatic profile with API®ZYM. Rhodotorula mucilaginosa was identified in both fish species. For the first time, Candida palmioleophila, Candida pseudorugosa, Cystobasidium slooffiae, and a member of the Yamadazyma genus were also identified and described as part of the normal fish gut–microbiota. Furthermore, the diverse enzymatic profile exhibited by some of these isolates suggests that it may be possible to develop novel applications for them, such as new probiotics and other biotechnological applications.