| The carbonate system(carbonate system)refers to a system relating the balance and the form transformations among different forms of inorganic carbon in water,which plays a very important role in the water environment. As one kind of large economic seaweeds which have a higher tolerance to temperature and salinity,Gracilaria tenuistipitata has become an important species for pond aquaculture in the coastal area of southeast China. Litopenaeus Vannamei occupies the head of the world’s three major shrimp species,having a wide range of salinity and alkalinity tolerance. In this study,in order to provide basic data for the tolerance of Litopenaeus Vannamei and Gracilaria tenuistipitata to different water carbonate system,the effects of different forms of inorganic carbon on the growth of Gracilaria tenuistipitata and Litopenaeus Vannamei were explored. The physiological traits of Gracilariatenuistipitata and Litopenaeus Vannamei under different inorganic carbon ratio were also monitored to analyze their adaptation mechanism in response of the environment changes. Moreover, the impacting pathways of different inorganic carbon on the algae and shrimp were investigated by means of inhibitors application.1. Under three CO2 concentrations(low,normal and high),a series of growth traits of Gracilaria tenuistipitata was measured,including specific growth rate,pigments contents,photosynthetic rates,chlorophyll fluorescence parameters. All of the traits were combined to imply the potential pathway and mechanism of CO2 utilization for Gracilaria tenuistipitata. The results showed that: With the increase of CO2 concentration,the growth of Gracilaria tenuistipitata increasing markedly. With the decline of the pigment content,the quantum yield of Gracilaria tenuistipitata increased. It is suggested that Gracilaria tenuistipitata could improve the CO2 utilization efficiency by changing its own physiological traits.2. Under three total alkalinity(12,24,and 36mmol/L)and five proportions of CO32-:HCO3-(0.125:1,0.25:1,0.5:1,0.75:1,1:1),the specific growth rate, pigment content,malondialdehyde(MDA)of Gracilaria tenuistipitatawere measured. The results showed that: When the CO32-:HCO3- ratio was less than 0.5:1,the specific growth rate was significantly higher than the control, indicating an appropriate alkalinity is beneficial to the growth of this algae. When the CO32-:HCO3- ratiowas more than 0.5:1,the specific growth rate decreased significantly with the increase of CO32-:HCO3-(P<0.05). It suggested that the increase of CO32-concentration in water could inhibit the growth of Gracilaria tenuistipitata,but the inhibition had nothing with total alkalinity. There were positive correlations between chlorophyll,phycoerythrin content and specific growth rate. Chlorophyll content decreased with the increase of CO32-:HCO3-,but phycoerythrin content didn’t change significantly(P>0.05). Compared with the control,the content of MDA under different ratio firstly increased and then decreased. It is suggested that Gracilaria tenuistipitata could adjust their pigment content and membrane lipid antioxidant system in response to CO32-: HCO3-increase.3. The photosynthetic oxygen evolution rate decreased with the increase of CO32-:HCO3-,indicating that the increase of CO32-concentration in water affected the photosynthesis of Gracilaria tenuistipitata. Three pathways of HCO3- utilization were inhibited by applications of carbonic an hydrase inhibitor acet Azolamide(Az),plasma membrane associated ATP enzyme inhibitor Vanadate(Van),Ⅲ-3 protein and ATP enzyme inhibitor4,4’- Diisothiocyanatostilbene- 2,2’- disulfonic acid, disodium salt(DIDS),respectively. The results showed thatunder total alkalinity of 12mmol/L,the inhibitory effect of Az and Van on the photosynthetic oxygen evolution rate was significant in all ratios(P<0.05),whereas the inhibitory effect of DIDS was not obvious. Under total alkalinity of 24mmol/L,the inhibitory effect of Az was significant in all ratios(P<0.05),the inhibitory effect of Van occurred in the lower ratios while the inhibitory effect of DIDS occurred in the higher ratios. Under total alkalinity of 36mmol/L,the inhibitory effect of Az and Van was significant in all ratios(P<0.05),while the inhibitory effect of DIDS only occurred in the higher ratios. It is suggested that,with the change of CO32-: HCO3- ratio,the HCO3-utilization pathway of this alga would change, and this change was associated with the total alkalinity.4. In an acute toxicity experiment, under the total alkalinity of 8mmol/L, Litopenaeus Vannameiwas exposed to different ratios of CO32-:HCO3-(0.125:1,0.25:1,0.50:1,0.75:1,1:1)to explore its tolerance to different carbonate concentration. The results showed that: During 24 h,the LC50 of Litopenaeus Vannamei occurred in the ratio of 1.53; During 48 h, the LC50 occurred in the ratio of 1.25; the 72 h LC50 ratio was 1.1 while the 96 h LC50 ratio was0.92. It is indicated that Litopenaeus Vannameihas a certain tolerance to CO32-increase in water,but the tolerance ability declines when the time goes on.5. For the purpose of exploring the lethal mechanism of different CO32-:HCO3-ratio on Litopenaeus Vannamei. Na+-K+-ATP enzyme inhibitor ouabain and carbonic anhydrase inhibitor acet Azolamidewere injected in this shrimp. After a 24 h period,the mortality of shrimp was recorded,and the changes of blood p H and p H ion transport activity were analyzed. The results showed that: There was no significant change of blood p H,which was maintaining at a relatively stable state. The activity of ion transport enzyme Na+-K+-ATP enzyme and HCO3--ATP enzyme decreased significantly when CO32-:HCO3- ratio was more than 0.5(P<0.05),the activity of V-ATP also increased,but the activity of carbonic anhydrase did not change markedly(P>0.05). It is indicated that the activity of p H ion transport enzyme is the main reason for stable blood p H. After the injection of ouabain,the mortality of shrimp increased significantly compared with the control(P<0.05),the mortality tends to be stable with the increase of CO32-:HCO3- ratio. This phenomenon ndicated that the elevated CO32-:HCO3- ratio was less lethal to shrimp when the Na+-K+-ATP enzyme was inhibited. However,the mortality of shrimp did not change significantly after injection of acet Azolamide(P>0.05) and had a similar change trend with the control. It is suggested that the Na+-K+-ATP enzyme of gill epithelial cells is one of the action sites of CO32-/HCO3-toxicity for Litopenaeus Vannamei.According to this study, either Gracilaria tenuistipitata or Litopenaeus Vannameihasa certain survival strategy for changes of water carbonate system. They can adapt to the changing external environmental stress through changes of physiological traits. However,when the stress exceeds a certain threshold,their survival and growth will be inhibited to some extent. |
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