Effects Of Elevated PCO₂ On The Survival And Growth Of Portunus Trituberculatus

Marine organisms are confronting unprecedented challenges from ocean acidification driven by human activity.Crustaceans will inevitably suffer from the effects of ocean acidification due to their sensitivity to seawater p H.Swimming crab Portunus trituberculatus is a crucial marine economic animal in China.To investigate the effect of elevated pCO₂on Portunus trituberculatus,we subjected swimming crabs to projected oceanic CO₂ levels（current:380μatm;2100:750μatm;2200:1500μatm）for four weeks,exposure to elevated pCO₂ mediated improved survival,but retarded the growth of swimming crabs.To reveal the main factors caused by these effects,in this study,crabs survival,growth,digestion,antioxidant capacity,immune function,tissue metabolites,gut bacteria of the crabs and on seawater bacteria were analyzed,and construct a structural equation model（SEM）,combined with seawater physical and chemical index and the function genes of seawater microbiome involved in carbon,nitrogen,phosphorus,and sulfur cycles.The main results of this thesis are as follows:1.In order to reveal the factors of retarded the growth of swimming crabs after exposure to elevated pCO₂,a nuclear magnetic resonance NMR-based metabolomics technique was used to analyze the changes of metabolic phenotypes of gut,muscle and hepatopancreas of swimming crab after exposure to elevated pCO₂.The results showed that exposure to elevated pCO₂ caused the elevated levels of amino acid and the accumulation of glucose and lactate in these three tissues.Overall,1500μatm pCO₂ caused a stronger effect than 750μatm pCO₂on the metabolite changes,as shown by an≈150% increase in tyrosine and tryptophan levels and a 73-86%increase in the levels of three branched chain amino acids and phenylalanine.In addition,the levels of muscle ADP and AMP fluctuated.The changes of metabolites in hepatopancreas was relatively small compared to those in gut and muscle,but exposure to elevated pCO₂ caused a 41.2-57.0% increase in the COS level,possibly due to acute hypoxia in hepatopancreas.Taken together,the inhibited aerobic glycolysis,energy oscillation,and enhanced protein and amino acid catabolism could partly explain the retarded growth of swimming crabs exposed to elevated pCO₂.2.In order to reveal the effects of elevated pCO₂ on the enzyme activities and oxidative stress and immune gene expression in hepatopancreas,the activities of digestive enzymes,antioxidant enzymes,and immune enzyme were measured s,as well as the levels of m RNA expression of antioxidant,stress,and immune genes.The results showed that exposure to elevated pCO₂ cause an increase in SOD and GST activity as well as MDA level,a decrease in ACP and AKP activity,and fluctuated in the levels of m RNA expression of antioxidant genes（c Mn SOD and ec Cu Zn SOD）,stress genes（Hsp70,MT2,and MBD4）,and immune genes（CTL2,g C1qR,and TRAF6）,indicated that elevated pCO₂ induced oxidative damage to the hepatopancreas and reduced immune capacity so that swimming crabs may be susceptibly infected by seawater external pathogens after exposure to a long-term acidification.The inhibited aerobic glycolysis,enhanced protein catabolism,and the decreased LPS activity may be related to decreased weight gain of swimming crabs.3.In order to reveal the effects of elevated pCO₂ on the gut and seawater bacterial community and their correlation,a 16S rRNA gene high-throughput sequencing technique was applied to analyze the changes of the structure and diversity of crab gut and seawater bacterial community after exposure to elevated pCO₂,and construct a correlation model based on SEM.The results showed that exposure to elevated pCO₂ had a small effect on the structure and diversity of crab gut and seawater bacterial community,elevated pCO₂ and the days may be major effects on the changes of the indicators of bacterial OTU.Based on SEM,carbonate system played a main effect in crab survival;seawater bacteria had a positive effect on crab survival and combined with gut bacteria and the metabolites of crab gut,muscle and hepatopancreas had positive effects on crab growth.4.In order to reveal carbonate system and seawater bacteria had major effects on crab survival,a tool for high-throughput quantitative assessment of microbial functional potential in carbon,nitrogen,phosphorus,and sulfur cycle was used to detect the changes in the absolute abundances of functional genes of seawater microbiome involved in carbon,nitrogen,phosphorus,and sulfur cycle and combined with the physical and chemical index of seawater to analyze the effects on crab survival.The results showed that ammonia excretion of swimming crabs after exposure to elevated pCO₂ may be resulted in increase in the concentration of NH₄⁺-N.Enhanced anaerobic ammonium oxidation and nitrification,and weakened nitrogen fixation,ammonification,and organic nitrogen mineralization caused decreased in the concentration of NH₄⁺-N.weakened denitrification and enhanced dissimilatory nitrogen reduction leaded to gradually increase in in the concentrations of TN,NO₂^--N,and NO₃^--N.The indicator of seawater bacterial OTU had significantly correlations with the physical and chemical index of seawater and the absolute abundances of functional genes of seawater microbiome involved nitrogen cycle respectively.Furthermore,the absolute abundances of functional genes of seawater microbiome were almost unchanged after exposure for 4 weeks,probably due to the bacteria’s adaptability to pCO₂.Based on SEM,the physical and chemical index of seawater had significantly effects on crab survival possibly due to the relatively low concentrations of NH₃,PO₄^3--P,and TP that reduced the toxicity to crabs and the probability of harmful algal blooms may be beneficial to crab survival in short term,but the accumulation of the concentration of NO₂^--N will have a negative effect on it.