Establishment Of A Cold Tolerance Zebrafish Model By Long-term Acclimation And Preliminary Functional Study

Temperature is an important physical and chemical properties in growth and survival of the organism.Change of the temperature which fishes live in has great influence in fish behavior,physiological and biochemical activities.The extreme temperatures will cause significant losses to the aquaculture industry.Therefore,establishment of cold tolerance fish model is beneficial to elucidate the gene regulatory networks and mechanism during low temperature.As an important fish model,many researches have been reported in zebrafish gene regulation under cold stress.Using zebrafish to stablish a fish model which growth under the low temperature,provide a convenient experimental material for researching cold tolerance behavior and mechanism in fish.Recently,it has been reported that organisms in the condition of less than extreme temperature will produce temperature acclimation effect to rise the ability of extreme temperature tolerance.In this research,we acquired a zebrafish line which is able to reproduce normally under lower temperature including the embryo critical temperature 22? and fatal temperature 20? by performing a long-term acclimation.In this study we performed one normal group TE27(The temperature 27?)and two acclimation group TA22(Acclimation temperature 22?),TA20(Acclimation temperature 20?)soring by raising temperature.Zebrafish enduring the acclimation in different low temperature shows normal abilities of growth and reproduction.The embryos produce by acclimation zebrafish line undergo normal embryonic development with 80% survival rate.Due to the low temperature of acclimation the hatch time of the offspring embryos extended.TA22 need 3-4 days to hatch,TA20 need longer of 5-6 days while the WT zebrafish need only 2.5-3 days.Further study on first generation offspring of the acclimation line,the embryos can normally growth to adult fish after embryogenesis in lower temperature without obvious difference with WT zebrafish.But the time for sexual maturity is longer than WT zebrafish,from 3-4 months to 6 months in 22? and 9 months in 20?.Important physiological indicator such as body size,reproducing activity,heart rate showed barely difference between the acclimation adult zebrafish and the WT zebrafish.The first generation offspring can survive in critical temperature 22? and fatal temperature 20? in embryo stage and reproduce normally in adult fish stage.Acclimation lower the critical temperature to 20? and fatal temperature to 18?and16?.The survival rate of TA20 group in 18? is 10%.We further investigate the cold stress adaptive mechanism of zebrafish using the acclimation line we mentioned before.Firstly we found the WT embryos can partly survive in fatal temperature 20? after develop in proper temperature 27? for a while.The cold stress mainly influent the embryogenesis stage and through observation and statistical analysis we locate the time point in blastula stage about 3.5hpf.Then we treat the acclimation group and WT group in 20? from fertilization to the postblastula stage.We further detected the expression of some gene related to DNA damage repair,cell growth,and cell cycle.gadd45?,C-jun,p21 showed significant upregulation in accumulation group.With the treatment of low temperature,apoptosis and ROS was detected in embryos in blastula stage.While the phenomenon reduced in acclimation groups,so we primarily predicted that the death of embryos in fatal temperature may related to DNA damage,and cell.More specific work will be taken to explore the mechanism.In order to know the reason why zebrafish embryos can survive in lower temperature after accumulation,we used both male and female zebrafish of TA22 and TE27 groups to perform different cross mate.We founded that the cold tolerance ability gains from female parent.We further detected the expression of mitochondria DNA and maternal miRNA in 0hpf stage,mitochondria DNA reduce in TA22 group,and one of the maternal miRNA(miR-202)upregulated in TA22 group.We infer that the cold tolerance ability shows after accumulation may gained from maternal factor.In this study we established a zebrafish model which can normally develop and reproduce in low temperature by long-term acclimation.The first generation offspring of accumulation group shows cold tolerance ability and the embryos can survive in lower temperature.We set a foundation in cultivate a cold tolerance zebrafish line,and provide a valuable biological model for research of zebrafish gene regulatory networks and mechanism during low temperature.