The Effect Of Crowding Stress And Nitrite Stress On Stress-Related Genes And Ubiquitin-Proteasome System In Grass Carp Ctenopharyngodon Idella

Fish,the aquatic and low-temperature vertebrate animal,has strong dependence on the external environment.The environmental factors can affect the survival of fish.Unreasonable farming patterns will cause the water environmental problems and the stress of fish.As a result,in the fish,the physiological function is disturbed,the drug resistance and immunity are reduced.And the unreasonable farming patterns also affect the quality of aquatic products,and even the death of fish.The ubiquitin-proteasome system(UPS)is an important system of concern for regulating protein degradation and function.It play important role in the cell cycle,apoptosis,metabolic regulation,immune response,signal transduction,transcription control,quality management,stress response,DNA repair and other biological processes.In this study,we investigated the effects of chronic crowding stress and acute nitrite stress on UPS activity,protein metabolism,stress response,and histology of muscles and liver tissues in the juvenile grass carp(Ctenopharyngodon idellu),with the purpose of promoting healthy aquaculture.In the first experiment,fishes were randomly split into low stocking density(0.9kg/m2)or high stocking density(5.9 kg/m2),white muscles were collected to assay.The expression of relative genes,ubiquitinated proteins,3-MH and Tyr were measured.In the second experiment,fishes were randomly exposed to three experimental groups(0,0.5and 20.0 mg/L),and samples of blood,liver,gill and muscle were collected at 12 h,24 h,48 h,96 h.The expression of genes,content of ubiquitinated protein,histological alterations of liver and muscle,and the levels of serum biochemical parameters of blood were investigated.The results are as follows:1.Fish exhibited significantly higher growth rate in the LSD group compared to the HSD group.There were no significant differences in the m RNA levels of nrf2,keap1,and hsp90 in white muscle of fish stocked at two densities at the endpoint of the experiment.In the UPS pathway,the expressions of ub,chip and psmc1 in the LSD group were significantly higher than those in the HSD group.Ubiquitinated protein level and the content of 3-Methylhiistiden elevated significantly in the LSD group.The m RNAlevels of mafbx,murf1,and s6k1 in the LSD group were significantly higher than those in HSD group.2.Compared to the control,serum cortisol levels significantly elevated at 12 h after exposure in a dose-dependent manner.And significant rise in serum cortisol level showed at 24 h and 48 h.Serum levels of alanine aminotransferase and aspartate aminotransferase were significantly decreased at 96 h.In the liver,the m RNA levels of nrf2 and hsp70 increased significantly at 12 h after exposure to 20.0 mg/L.Nitrite exposure of 20.0 mg/L caused the significant elevations in the expressions of chip at 12 h,24 h and 48 h,and there was no significantly difference at 96 h between treatment groups and control group.The expression levels of ub increased with a dose-dependent increase at different time under nitrite stress.In the 20.0 mg/L exposure group,the m RNA levels of psma2 and psmc1 increased significantly at 24 h,decreased significantly at 48 h,and returned to the control levels at 96 h.After exposure of nitrite for 12 h,the content of ubiquitinated proteins in the liver increased significantly,and remained significantly higher than that those in the control groups at 96 h.Hepatic vacuolization was observed in the liver of grass carp at 96 h after exposure to the concentration of nitrite(20.0 mg/L).In the muscle,at 12 h and 24 h,the expression levels of hsp70 was significantly lower in the nitrite exposure group than those in the control group,and at 48 h,its expression level showed significant increase.The expression levels of ub were significantly higher in exposure group than those in control group at 96 h.Under nitrite stress,the expressions of psma2 and psmc1 were significantly decreased at 12 h,24 h and 48 h.At the 0.5 mg/L exposure group,the expressions of murf1 and mafbx increased significantly at 12 h,24 h,and 96 h.The expression levels of ubiquitinated proteins in the dorsal muscles increased significantly at 24 h of stress,and there were no obvious difference among groups at 48 h and 96 h.The histological changes of muscle were not found at 96 h of 20 mg/L.After the exposure of 20 mg/L nitrite at 96 h,the gill showed obvious aneurism.In conclusion,the stress-related parameters showed that grass carp appeared to be adapted to the crowding environment after 70 days.The fish exhibited slower growth rate and the down-regulation of UPS in the high stocking density group,while both proteinsynthesis and degradation manifested high activity levels in the low stocking density group.High concentrations of nitrite stress caused the stress response of grass carp,significantly increased the expression of hsp70,nrf2 of liver,and resulted in the damage of liver and gill.Moreover,the ubiquitin-proteasome system of liver and muscle were activated to remove damage or false proteins to reduce the oxidative damage risk.