Impact Of Stress Hormones On Iron Homeostasis Of Cyprinid Megalobrama Amblycephala And Pathogenicity Of Pathogenic Bactaeria Aeromonas Hydrophila

Stress response is one of the important inducing factors in outbreak of aquatic animal bacteriosis.The hormone levels,metabolism and immunity of the organism was dramatically changed under stress status.Iron is an important multi-bioactive trace element in fish and micro-organisms,engaged in diverse metabolism process,like oxygen transport,cell proliferation,electron transfer.And iron is an indispensable nutritional factor which is contended after the host infected by pathogenic bacteria.Current research on fish immune function stress suppressed by stress have been reported,however detail mechanisms upon invading the host by pathogens remains unknown.From the perspective of the stress hormones,we investigated changes of iron levels and related regulatory genes expression(hepcidin and transferrin)after acute stress,as well as impact of stress hormones on pathogenicity of pathogenic bacteria.This study is hoping to provide basic information for etiology and pathology of fish stress.Megalobrama amblycephala is a large-scale breeding freshwater fish,which met serious challenges stress response and outbreak of bacterial hemorrhagic disease caused by Aeromonas hydrophila during the culture process.After simulating acute stress response in M.amblycephala by injecting cortisol,we detected iron content in plasma and liver,and determined the expression of hepcidin and transferrin gene by quantitative PCR at different time points.We also simulated the environment in vivo by using iron-deficiency serum-SAPI medium,and made comprehensive survey of changes in microbial growth,expression of virulence related genes,phenotypes and virulence in microbes treated with stress hormone.The consequences are as follows:1.After cortisol injection(10 mg/kg),M.amblycephala absorbed celiac cortisol at short notice,and caused cortisol concentration in plasma is the highest at 0 hour post injection(hpi).plasma cortisol of experimental group thereafter faded away gradually at 2,4,8,10 hpi,but maintained a higher concentration of plasma cortisol(P<0.05)until concentration of plasma cortisol was consistent with control group at 12 hpi.These results suggested cortisol injection is adapted to establish Chinese bream acute stress model.Content of plasma iron in experimental group had no significant change within 0-2hpi,and accumulated continually after 4hpi.There was a maximum at 8hpi,and decreased to control level.In liver,iron content produced a curve shaped like V,first maintained stable within 0-2hpi,then decreased sharply at 4hpi,and appeared a low valley at 10 hpi due to iron loss,eventually recovered to control level after 24 hpi.In liver,gene expression of LEAP1,LEAP2 and Tf showed a unimodal change trend,and had the highest expression levels of these genes at 8,10,10 hpi,respectively,then restored to normal genes expression at 24,48,48 hpi,respectively.Levels of Tf gene expression in intestine increased slightly at 0hpi,and began to transcribe heavily within 8 hpi until Tf gene expression level restores to normal level at 48 hpi.These results implied that iron homeostasis disruption in M.amblycephala which caused by cortisol injection,induced expression of iron homeostasis associated genes,and then activated iron regulation system in living beings.2.The growth curve of A.hydrophila AH196 shaped like a normal S in serum-SAPI medium.Higher concentrations of norepinephrine(NE)(100μM and 200μM)can make A.hydrophila AH196 proliferate faster in exponential growth period,but low concentrations of NE(12.5,25 and 50μM)did not enhance micro-organisms growth.NE at the concentration of 100μM could induce the growth of A.hydrophila NJ-35,AH33,AH189 and AH301.Quantitative PCR results showed,A.hydrophila treated with NE highly upregulated the expression levels of heat-stable enterotoxin(ast),mRNA expression of hemolysin(hly),iron superoxide dismutase(sodB)and flagellin B(flaB)gene(P<0.05).Exposure to NE dramatically increased the expression of outer membrane protein W(omp W),outer membrane protein A(ompA),serine protease(ahp),elastase(ela),adhesin(aha),iron uptake gene regulator(fur)and ahy R gene(P<0.01).NE couldn’t alter mRNA expression of aerolysin(aerA),heat-labile enterotoxin(alt)and lipase(lip)gene,but significantly inhibited the cytotoxic enterotoxin(act)and flagellin protein A(flaA)mRNA expression.NE only enhanced A.hydrophila AH196 protease activity,but exercised on significant effect on motility and the activity of hemolysis and lipase activity.In vivo challenge tests exhibited,NE observably increased the mortality of Carassius auratus after infected with A.hydrophila,so that NE enhanced the virulence of A.hydrophila.