Zebrafish (Danio rerio) larvae as a system to test the efficacy of polysaccharides as immunostimulants

The present study was carried out to examine the use of zebrafish (Danio rerio) as a preliminary screening model for testing the effect of potential immunostimulant substances on the innate immune system. β-Glucan, a polysaccharide used widely as an immunostimulant, was used as a representative molecule and tested on zebrafish embryos and larvae. The efficacy of the molecule was evaluated by determining the differential expression of some selected genes related to the immune system by RT-qPCR. Larvae from 72 hours post fertilization were found at the optimal developmental stage for assessing the expression of the selected genes. To verify if the β-glucan entered the larvae and therefore was responsible for the effects produced, the molecule was labeled fluorescently to check its localization by using microscopy. For estimating the effects of β-glucan on gene expression, zebrafish embryos and larvae were immersed in three different concentrations of β-glucan (50, 100, and 150 μg/mL) using five different exposure times. A stronger gene induction was observed when longer times of exposure and older larvae were used. The most evident effects of β-glucan were the overexpression of the genes TNFα, MPO, TRF, and LYZ. Moreover, slight changes in MPO expression were detected using a transgenic line of zebrafish (MPO::GFP), and a temporal increase in resistance against Vibrio anguillarum was found after β-glucan immersion. The assay used in this study permits the testing potential of immunostimulants in a simple and cost-effective way.