Effects Of Calcium And Magnesium On Development Of Larvae Of Exopalaemon Carinicauda And The Study Of Domestication With The Low Saline-alkaline Water

There are 99.15 million hm² of saline-alkali land in China,among which there are 6.667 million hm²of saline-alkali land along the Yellow River.Fishery along the Yellow River saline-alkali land has been dominated by fish culture for a long time,with single culture mode,high culture density,increasingly serious breeding diseases,increasing pollution,coupled with the lack of low-saline-alkali ecological adaptive breeding varieties,the development of aquaculture along the Yellow River has encountered a bottleneck.Exopalaemon carinicauda is a representative species of Palaemonidae.It has delicate meat,delicious flavor,fast growth rate,low salt tolerance and other advantages.It can grow normally in water with a salinity of 4-35 and can survive in pure fresh water through gradual desalination,and it is a good variety that can be transplanted and domesticated in inland low salt alkali water.This study aims to clarify the water quality characteristics along the Yellow River saline-alkali waters in Henan province,develop the saline-alkali water acclimation technology according to different regions and water quality characteristics,and explore the effects of low saline-alkali conditions and different ratios of calcium and magnesium on the metamorphosis development,antioxidant enzyme activity,osmotic pressure regulation related enzymes and gene expression,so as to provide theoretical basis and technical support for the aquaculture of E.carinicauda along the yellow saline-alkali waters.The main research results are as follows:1.Investigation and analysis of the characteristics of salt and alkali water quality along the Yellow Riverin Henan ProvinceIn this chapter,the characteristics of water quality of wells and ponds in Zhengzhou,Xinxiang,Kaifeng and Puyang along the Yellow River in Henan Province were studied,and the p H,salinity,hardness,Na⁺,K⁺content and other indexes were measured.The results showed that the p H value,salinity,hardness and alkalinity of the water samples from the four collection points in the Yellow River of Henan Province ranged from 6.96 to 9.07,from 0.5 to 2.73,from 4.36 to 22.34mmol/L,and from 4.6 to 13.64mmol/L.The alkalinity of the water samples was mainly composed of HCO₃^-.The main cation was Ca²⁺,Mg²⁺and Na⁺,and the content of K⁺was low.2.Effects of different salinity and calcium and magnesium ratios on the metamorphosis development oflarva E.carinicaudaIn this chapter,the effects of different ratios of Ca²⁺and Mg²⁺on the metamorphosis development of E.carinicauda were studied at salinity of 15,10,5 and 5.The results showed that in the 14th day,all the15 and 10 salinity groups became larvae;in the 15th day,all the five salinity groups became larvae.The survival rates were 71.00%,74.00%and 78.56%in the groups of 15,10 and 5 salinity,respectively.When the calcium concentration was 50 mg/L at the salinity of 5,all larvae in groups 1:1.5 and 1:2.5 changed into larvae in the 12th day,all larvae in groups 1:3 changed into larvae in the 13th day,and all larvae in groups 1:1 and 1:2 changed into larvae on the 14th day.The survival rates of 1:1,1:1.5,1:2,1:2.5 and 1:3groups were 18.75%,65.00%,56.25%,60.00%and 67.50%,respectively.When the calcium ion concentration was 80 mg/L,all of the 1:2.5 groups changed into larvae on the 12th day,all of the 1:1.5 and1:3 groups changed into larvae on the 13th day,and all 1:1 group changed into larvae on the 14th day.The survival rates of 1:1,1:1.5,1:2,1:2.5 and 1:3 groups were 28.09%,66.25%,58.75%,78.56%and 63.33%,respectively.3.Study on low salinity and alkali acclimation technology of larvae of E.carinicaudaIn this chapter,low salinity and alkali acclimation were carried out on the larvae of E.carinicauda.Firstly,the salinity was reduced day by day according to the salinity gradient of 10→7→5→3→2→1,10→5→3→2→1,5→2→1,5→1,and 5→1.After 3 days of stability in salinity 1 water,the alkalinity was increased day by day according to the alkalinity gradient of 2→4→5→6,2→6 mmol/L carbonate.The results showed that the survival rate of larvae was 100%after 72 hours of low salinity and alkali acclimation.4.Effects of calcium and magnesium ratio on growth and enzyme activity of juvenile E.carinicaudaunder different calcium ion concentrationsIn this experiment,30,40 and 50mg/L calcium concentration groups were set,and the ratios of calcium and magnesium at each concentration were 1:1,1:1.5,1:2,1:2.5 and 1:3,respectively.The larvae of P.SPP were cultured in the above water bodies for 4 weeks.After the end of culture,the survival rate and weight gain rate were counted,and the activities of T-SOD,CAT,GSH-Px,T-AOC,AKP,ACP,Na⁺-K⁺-ATP and Ca²⁺-ATPase were determined.The results showed that the survival rate and weight gain rate were all higher than 70.0%and 397%in all experimental groups.When the calcium concentration was30,40 and 50mg/L,the activity of T-SOD enzyme was the highest when the ratio of Ca²⁺and Mg²⁺was1:2-1:2.5.When the calcium ion concentration was 30mg/L,the CAT enzyme activity was the highest when the calcium and magnesium ratio was 1:2;when the calcium and magnesium ratio was 40mg/L,the CAT enzyme activity was the highest when the calcium and magnesium ratio was 1:2.5.When the calcium ion concentration was 30mg/L,the GSH-Px activity was the highest when the calcium and magnesium ratio was 1:3,and there was no significant difference compared with the 1:2 group（P>0.05）.When the concentration of calcium ions was 40 and 50mg/L,the activity was the highest when the ratio of calcium and magnesium was 1:2.T-AOC activity was the highest when Ca²⁺concentration was 30,50mg/L and Ca/Mg ratio was 1:1.5.When the concentration of calcium ions was 40mg/L,the highest activity was found when the ratio of calcium and magnesium was 1:2.When the concentration of calcium ion was30mg/L,the activity of ACP and AKP was the highest when the ratio of calcium to magnesium was 1:1.5;ACP and AKP activities were the highest when the concentration of calcium ion was 40 mg/L and the ratio of calcium to magnesium was 1:2;when the concentration of Ca²⁺was 50mg/L,the ratio of Ca²⁺to Mg²⁺was 1:2.5,the AKP activity was the highest;when the ratio of calcium and magnesium was 1:2,ACP activity was the highest.When Ca²⁺concentration was 30,40 and 50mg/L,Na⁺-K⁺-ATPase activity was the lowest at Ca/Mg ratio of 1:2（P<0.05）,and Ca²⁺-ATPase activity was the highest at Ca/Mg ratio of 1:3,1:1.5 and 1.1（P<0.05）,respectively.5.Effects of Carbonate Alkalinity Stress on the Expression of V-H⁺-ATPase and Na⁺-K⁺-ATPase in E.carinicauda under Low SalinityIn this experiment,V-H⁺-ATPase gene was obtained by RACE technology,and its bioinformatics was analyzed.The expression levels of V-H⁺-ATPase and Na⁺-K⁺-ATPase in gills and hepatopancreas were quantitatively determined by fluorescence at 3,6,12,24,48,72h and 8 weeks after carbonate alkalinization stress.The results showed that the full-length c DNA of V-H⁺-ATPase gene was 2405bp,with an open reading frame（ORF）of 1470bp,a 5’non-coding region of 32bp,a 3’non-coding region of 902bp,and encoded 489 amino acids in total.Amino acid sequence alignment showed that V-H⁺-ATPase was more than 95%consistent with other crustaceans in evolution,and was the closest relative to Penaeus vannamei.V-H⁺-ATPase gene was expressed in all tissues of E.carinicauda,and the highest expression was found in gill,followed by hepatopancreas.The relative expression of V-H⁺-ATPase in gills of the stress group was significantly higher than that of the control group under carbonate alkalinity stress for 6,12,72h and 8 weeks（P<0.05）.After 72h of stress,the Na⁺-K⁺-ATPase gene in gills of stress group was significantly higher than that of control group（P<0.05）,but there was no significant difference after 8weeks（P>0.05）.The expression level of V-H⁺-ATPase in hepatopancreas in stress group was significantly higher than that in control group at 3,6h and 8 weeks（P<0.05）.After 3-72h of stress,the hepatopancreas Na⁺-K⁺-ATPase expression level in stress group was significantly higher than that in control group（P<0.05）,and there was no significant difference between stress group and control group after 8 weeks（P>0.05）.