Comparative Studies On Ammonia-N Related Enzymes And Corresponding Genes Responding To Ammonia Between Different Families Of Shrimp Litopenaeus Vannamei

Shrimp(L.vannamei)has good adaptability,fast growth rate and high meat quality.L.vannamei was introduced into China since 1988,and it has become one of the world's dominant species in shrimp farming in recent 30 years.However,with the increased degree of intensive farming and discharge of sewage,contaminants have accumulated severely and ruined farm water environment seriously.This situation has brought serious economic losses in the aquaculture industry.As a common environmental pollutant in water,ammonia nitrogen(ammonia-N)could penetrate into the tissue fluid by osmosis and causes ammonia poisoning in L.vannamei.Studies show that ammonia-N might influence immune system,growing rates and osmotic pressure regulation in L.vannamei.L.vannamei with different stages shows varied sensitivity to ammonia-N stress,and shrimps in different families also show varied sensitivity among the same size of shrimps.This study explores the toxicological effects of ammonia-N to different stages of L.vannamei systematically.Meantime,the families with poor tolerance to environment(strain A 3281)and strong resistance(strain B 3271)were selected in this study.The purpose of this work is to evaluate the ammonia-N(NH4+)metabolic regularity by testing the related enzyme activities(GDHase,GSase and TGase)and gene expression(GDH-?GS and TG)between two L.vannamei strains under ambient ammonia-N tolerance(3.4,13.8 and 24.6 mg/L).We hope to reveal the mechanism of stress response of different strains of L.vannamei and lay the foundation for selecting new species with high resistance to ammonia-N.The main results are as follows:1.In the early growth stage,Zoea ?(Z 2)of L.vannamei is the most sensitive stage to ammonia-N stress.The sensitivity to ammonia-N in L.vannamei is quite different in different growth stages;compared to larvae stage,the adult shrimps have a higher tolerance to ammonia-N.Z 2 stage is the most sensitive stage to ammonia-N among the stages from Nauplius to Postlarvae.The median lethal concentration(LC50)of ammonia-N is 17.811 mg/L in the stage Z 2,and it can be used as a target stage to detect ammonia-N stress.2.The temporal and spatial variation of three enzymes related to ammonia-N metabolism.(1)Comparative analysis of GDHase activity in different tissues of two strainsAmmonia-N stress(3.4-24.6 mg/L)significantly affected the activities of GDHase in two strains of L.vannamei.The activity of GDHase in muscles from both strains showed a decreasing tendency with the increase of ammonia-N stess.In hepatopancreas,the activity of GDHase in both stains showed a increasing tendency within five days,while a decreasing trend was present from five to ten days.The change of GDHase activity in two tissues was consistent between two strains(p>0.05).(2)Comparative analysis of GSase activity in different tissues of two strainsIn general,with the increase of ammonia-N stress,the activity of GSase in muscles of two strains was increased.However,the trend appeared to be different between two strains in hepatopancreas.In strain A,the activity of hepatic GSase was inhibited with the increase of ammonia-N concentration.In strain B,GSase activity was only increased on the first day then it was inhibited in hepatopancreas(T>5 days).(3)Comparative analysis of TGase activity in different tissues of two strainsAs to the activity of TGase,a short time of ammonia-N stress(T<5 days)could significantly inhibit the activity of TGase in two tissues from both strains(p<0.05).However,the trend appears different between two strains.Compared to the control,the activity of TGase in two tissues of strains B was still inhibited from 5 to 10 days,while it was activated in both tissues of strains A.We can infer from data of these ammonia related enzyme activity,to deal with the ammonia-N stress,muscle in two strains of L.vannamei can activate activities of catabolic enzyme and inhibit productive enzyme activity to maintain ammonia balance in the organization;3.The analysis of the integrated biomarker response(IBR)index of three enzymes related to ammonia-N metabolismThe IBR index showed that different concentrations of ammonia-N stress can significantly affect GSase and TGase activity in muscles from two strains.Meantime,the influence increased with the increase of ambient ammonia-N stress.The reaction time of strains B(T<5 days)was significantly earlier than that of strains A(5<T<10 days).We can deduce that muscle could convert glutamate and NH4+ into glutamine to metabolize excess NH4+ when ambient ammonia-N stress increased.The data of IBR also demonstrated that the stress was up-regulated in hepatopancreas while it decreased in muscle under raised ammonia-N concentration(3.4-24.6 mg/L).This data suggests that the hepatic tissue might be the main tissue which causes the responding difference between two strains.Strain B could stimulate the activity of ammonia metabolizing enzymes in earlier stage,which played an important role in maintaining the stability of the content of ammonia-N in the shrimp.4.The temporal and spatial variation rules of related genes expression response to ammonia-N stress(1)Comparative analysis of related gene expression to ammonia-N stress in muscles from two familiesAmmonia-N stress increased the GDH-? and GS expression in muscles of two strains significantly(p<0.05).The expression level in strain B was significantly higher than that of strain A.The results showed that strain B had a stronger ability to transform ammonia-N,which reveals the reasons of the tolerance difference between strains in the gene level.The expression of TG gene between two strains tends to be opposite in hepatopancreas after five days.The expression of TG gene in strain A was significantly down regulated,while it was up-regulated in strain B with the increased ammonia stress.(2)Comparative analysis of related gene expression to ammonia-N stress in hepatopancreas of two strainsCompared to the control,ammonia-N stress could significantly inhibit the expression of GDH-?,GS and TG gene in hepatopancreas of strain A in five days(p<0.05).While,the expression of these genes in hepatopancreas of strain B was activated by increased ammonia-N stress.The hepatic GDH-? and GS gene were up-regulated in two strains from five to ten days.The TG gene in hepatopancreas of two strains showed the same depressed trend with the increase of ammonia-N stress.The results showed that the L.vannamei with high adaptability could adapt to ammonia stress by activating ammonia decomposed gene expression(GDH-? and GS)as well as inhibiting ammonia synthesis gene expression(TG).This study provides useful data of L.vannamei under ammonia-N stress by testing related enzyme activities and gene expressions.At the same time,this work might help to reveal the mechanism of ammonia-N in diverse strains and speed up the breeding process of new species in L.vannamei.