The probiotics Vibrio diabolicus (Ili), Vibrio hepatarius (P62), and Bacillus cereus sensu stricto (P64) colonize internal and external surfaces of Penaeus vannamei shrimp larvae and protect it against Vibrio parahaemolyticus

Abstract

The vibrios carrying the pirA and pirB toxin genes cause acute hepatopancreatic necrosis disease (AHPND) in cultivated shrimp, significantly affecting the yield. The use of probiotic strains is a promising strategy to prevent vibriosis in shrimp cultures. The microbial consortium formulated with three strains, Vibrio diabolicus (Ili strain), Vibrio hepatarius (P62 strain), and Bacillus cereus sensu stricto (P64 strain), is used to control vibriosis. Despite the effectiveness of these probiotics, until now there is not a description of the cellular structures necessary to help shrimp colonization, an important process to exclude pathogenic vibrios. Initially, the three probiotic strains were characterized by scanning electron microscopy, which allowed us to observe that Ili has numerous flagella and P62 has numerous fimbriae, while P64 has both flagella and fimbriae. Next, the colonization capacity of the three probiotic strains in P. vannamei shrimp larvae was determined by direct exposure of the shrimp larvae to probiotics previously stained with fluorochrome dyes, orange acridine, Evans blue, and DAPI. The larvae were observed using epifluorescence and confocal microscopy, which showed the three probiotics adhered to the body cuticle and internal gut lining. Larvae exposed to AHPND-causing V. parahaemolyticus stained with acridine orange were quickly and completely colonized, however, the previous treatment of larvae with probiotic consortium limited the colonization of the pathogenic Vibrio. The ability of probiotic strains to exclude pathogenic vibrio from shrimp surfaces could be explained by the cell surface appendages observed in probiotic strains, thus limiting the free spaces for pathogenic vibrios. Finally, the probiotic consortium in P. vannamei larvae was evaluated in a larviculture (Nauplii 5 to postlarvae 8), where the three probiotics were applied daily. The trial included a control group without the application of the probiotic. After 16 days of culture, the larvae treated with the microbial consortium and the control group were challenged with a pathogenic, the AHPND-causing V. parahaemolyticus strain. We registered a significant (P < 0.05) higher survival in the larvae treated with the microbial consortium 75.33 ± 12.62% compared to the control group 32.27 ± 9.57%. These results indicate that the preventive use of probiotic strains with colonization capacity increase larvae protection. This may be a good strategy for the control of AHPND in shrimp larvae hatcheries.