Impact of Pronase, Sodium Thiosulfate, and Methylene Blue Combinations on Development and Survival of Sodium Hypochlorite Surface-Disinfected Zebrafish (Danio rerio) Embryos

Abstract

Embryo surface disinfection is utilized in aquaculture to decrease the risk of pathogen introduction into established colonies. Zebrafish embryos are commonly disinfected with unbuffered sodium hypochlorite at 25-50 ppm for 10 min with or without concurrent treatment with chemicals, including pronase (Pron), sodium thiosulfate, and/or methylene blue; however, the impact of these chemicals on embryo survival and development has not been evaluated. In this study, AB and casper embryos were exposed to disinfection protocols that used Pron, sodium thiosulfate, and/or methylene blue (given alone, in various combinations, or all three combined) with 50 and 100 ppm sodium hypochlorite performed 6 and 24 h postfertilization (HPF). All groups were evaluated for survival, hatching, and malformations at 5 days postfertilization. Maximal survival (69%-97%) and hatching rates (66%-94%) were generally observed with sodium hypochlorite disinfection followed by exposure to both Pron and sodium thiosulfate and maintenance in standard embryo medium without methylene blue. Methylene blue had variable effects on survival and hatching. Higher survival and hatching rates were seen in AB embryos disinfected at 6 HPF and casper embryos disinfected at 24 HPF. Susceptibility to sodium hypochlorite toxicity differed by strain, emphasizing the need to test disinfection protocols on small embryo cohorts.

Challenging the status quo: Methylene blue at recommended concentrations alters metabolism in early zebrafish development

“…Finally, we also observed that the CSH concentration (0.0001%) of MB induces early hatching. Previous studies reported conflicting results regarding the effect of MB exposure on hatching 43,44,57 . Because chemical exposure-dependent alterations in hatching time have been reported as endpoint in zebrafish toxicity testing 65 , future studies should investigate possible interactive effects of MB and test chemicals on this endpoint in toxicity screening studies.…”

“…Despite the long-known metabolic effects of MB, it remains unknown whether currently recommended MB concentrations affect early zebrafish metabolism, and thus may act as a widespread confound in experimental approaches using early zebrafish. Indeed, possible effects of MB on zebrafish (eleuthero)embryos have only very recently begun to be investigated [43][44][45][46][47] . The few studies published to-date focused on overt developmental and behavioural endpoints but yielded conflicting results.…”

The Potentials of Methylene Blue as an Anti-Aging Drug

“…Additionally, MB was shown to photo-inactivate bacteria in 1928 [ 20 ], and its potent antiviral effects were discovered soon after [ 21 ]. MB can also be used as an anti-fungal and anti-parasitic aquarium disinfectant, even with very high dosages (millimolar) [ 22 , 23 ]. MB has also been used in photodynamic therapy among different types of cancers, including lung cancer [ 24 ], breast cancer [ 25 ], and prostate cancer [ 26 ].…”