Protective Yeasts Control V. anguillarum Pathogenicity and Modulate the Innate Immune Response of Challenged Zebrafish (Danio rerio) Larvae

Abstract

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.

The Intestinal Mycobiota in Wild Zebrafish Comprises Mainly Dothideomycetes While Saccharomycetes Predominate in Their Laboratory-Reared Counterparts

“…In addition, these fungi are involved in host defense in fish. Zebrafish larvae pre-colonized with several yeast strains showed an improved immune response against the pathogen Vibrio anguillarum and increased survival compared to non-colonized larvae ( Caruffo et al, 2015 , 2016 ). Some yeast can also influence the establishment of other gut microbes in fish, thus influencing host immunity ( Gatesoupe, 2007 ; Vohra et al, 2016 ).…”

“…Ascomycota yeast strains Debaromyces and Saccharomyces isolated from rainbow trout ( Oncorhynchus mykiss ) have a strong adhesion potential to fish intestinal mucus and may compete with other microorganisms, mostly bacteria, within the gastrointestinal tract ( Vázquez-Juárez et al, 1997 ; Andlid et al, 1998 ). Ascomycota fungi are also involved in stimulation of the fish immune system and protection against pathogenic bacteria ( Caruffo et al, 2016 ). Basidiomycota occupied a minor proportion of the zebrafish intestinal fungal community - considerably higher level in the laboratory samples (Bodø and Uttara) compared to the wild-caught fish ( Figure 1A ).…”

Gnotobiotic rainbow trout (Oncorhynchus mykiss) model reveals endogenous bacteria that protect againstFlavobacterium columnareinfection

“…To improve evidence-based identification of fish probiotics and their efficacy in disease prevention, the use of germ-free (GF) or fully controlled gnotobiotic hosts is a promising strategy [20, 21]. In addition to laboratory fish models such as zebrafish ( Danio rerio ) [22-24], several fish species have been successfully reared under sterile conditions to test probiotic-based protection against pathogenic bacteria, including Atlantic cod ( Gadus morhua ) [25], Atlantic halibut ( Hippoglossus hippoglossus ) [26], European sea bass ( Dicentrarchus labrax ) [19] and turbot ( Scophthalmus maximus ) [27] (for a review, see [28]).…”

Peer Review #1 of "Systematic evaluation of the gut microbiome of swamp eel (Monopterus albus) by 16S rRNA gene sequencing (v0.2)"

“…Many studies have revealed that the administration of probiotics could modulate gut microbial balance, enhance immune status, reduce disease susceptibility and improve feed efficiency (Caruffo et al, 2016;Hai, 2015;Newaj-Fyzul & Austin, 2015). It suggested that the dysbiosis and homeostasis of gut microbiota might be associated with swamp eel's diseases pathogenesis and food digestion.…”