Immune Response Of MiR-23a And Susceptibility To High Temperature And Hypoxia In Grass Carp Infected With Aeromonas Hydrophila

Grass carp（Ctenopharyngodon idella）is the most productive aquaculture fish in the world.With the development of grass carp culture,diseases have occurred from time to time,and the disease caused by Aeromonas hydrophila infection caused serious economic losses.In this study,the target genes of mi R-23a were screened and determined to investigate the mechanism of mi R-23a after A.hydrophila infection,and explored the effects of high temperature and hypoxia as combined stressors on grass carp.The main findings are as follows:21 miRNAs were abnormally expressed in the spleen of resistant and susceptible groups after grass carp infection with this bacterium,indicating that mi RNAs might be involved and play important functions in the process of bacterial infection of grass carp.The expression of mi R-23a changed significantly at different time points in（Ctenopharyngodon idella kideny,CIK）CIK cells infected by A.hydrophila.In the dual-luciferase reporter assay and experiments of mi R-23a agomir/antagomir transfection,the target genes were reversely regulated,and TBK1,GLUT1 were identified as the target genes of mi R-23a.The expression of ldha、ldhba、pdha1a and pdha1b were suppressed after overexpression of mi R-23a.These results showed that mi R-23a was involved in the regulation of immune response in CIK cells after A.hydrophila infection.TBK1 and GLUT1 are the target genes of mi R-23a.mi R-23a can affect the expression of downstream genes by targeting GLUT1.Climate change will have a profound influence on the spread and development of parasites and diseases in aquatic ecosystems.A.hydrophila,a common causative agent of bacterial septicemia in freshwater fish,has a significant mortality rate in the grass carp aquaculture.In this experiment,the cumulative mortality and bacterial load（gill,intestine and liver）were detected,the activities of immune enzymes and inflammatory factors in the liver of grass carp were studied,and the histological changes of liver after infection with A.hydrophila were quantified by semi-quantitative system to investigate whether high temperature and hypoxia as combined stressors have potential positive effects on grass carp in the process of A.hydrophila infection.High temperature and hypoxia resulted in increased bacterial load in the gill(7.02×10⁶ vs.4.39×10⁶ CFU g^-1 on 1dpi),intestine(6.17×10⁸vs.1.14×10⁸ CFU g^-1 on 3dpi),and liver(5.72×10⁴vs.0 CFU g^-1 on 2dpi),which may create favorable environment for pathogen invasion,dissemination and multiplication into tissues,leading to higher mortality rate（11.67±3.33%vs.3.33±3.33%）.Furthermore,under high temperature and hypoxia,the acetylcholine activity and inflammatory cytokine expression in the liver（interleukin 1β,interleukin 6,interleukin 8 and interleukin 10）were the highest on 1 dpi;the adenosine deaminase,alkaline phosphomonoesterase,superoxide dismutase,and catalase activities in the liver were significantly reduced on 1 dpi.Our results showed that the liver index under high temperature and hypoxia was higher than that of the normal controls on 5 dpi.The negative impact of A.hydrophila infection were aggravated under the condition of high temperature and hypoxia.The experimental results suggested that grass carp disease increased upon exposure to high temperature and hypoxia together with A.hydrophila.