Monitoring And Evaluation Of Biomass And Biogenic Elements Of Exaiptasia Diaphana In The Dapeng Peninsula Sea Area Of Shenzhen

The Exaiptasia diaphana,is usually reported to be distributed in the North Atlantic coast,the Caribbean Sea and the North Pacific region.In recent years,it has gradually been found in the eastern waters of Shenzhen,China,and has a tendency to spread in an outbreak.The Exaiptasia diaphana will occupy the habitat of other organisms,and have an impact on the marine environment.In particular,adhering to the net cage will cause the net clothing of the net cage to be blocked,and the exchange of seawater inside and outside the net clothing will become worse,leading to the occurrence of diseases of breeding species and other hazards.In addition,the Exaiptasia diaphana is also considered as a pest in the aquarium industry.At present,the research on Exaiptasia diaphana is mainly focused on using it as a coral model system to study the cell,physiological,ecological characteristics and albinism mechanism of the symbiosis of echinococcus and dinoflagellate.However,Exaiptasia diaphana is an alien species to China,and has not been effectively monitored and studied for its biogenic elements.In this paper,Exaiptasia diaphana was collected from Shenzhen sea area as the research object,carried out molecular identification,compared the common sea anemone and the close relatives of Exaiptasia diaphana in Shenzhen sea area,designed a method to detect Exaiptasia diaphana based on environmental DNA,and optimized the determination method of total phosphorus in the tail water of aquaculture into the sea,in order to improve the detection efficiency of total phosphorus in the culture water of Exaiptasia diaphana,By comparing the effects of different concentrations of phosphate on the physiological and biochemical characteristics of Exaiptasia diaphana,the reason for the high density of Exaiptasia diaphana in Shatanhai Bay,Shenzhen was preliminarily analyzed.The results are as follows:The specific primers and TaqMan probes designed for the Cytb gene only have positive amplification on the samples of Exaiptasia diaphana.The limit of detection（LOD）and the limit of quantification（LOQ）are 25.33 copies·reaction^-1 and 138.00copies·reaction^-1,respectively.The slope of the standard curve is-3.24,the amplification efficiency is 103.4%,and the correlation coefficient（R²=0.995）.In the simulated water environment,there is a linear positive correlation between the number of Exaiptasia diaphana（y）and the concentration of eDNA（x）,y=0.0002x±3.8947（R²=0.930）,but the fitting equation at the lower density is y=0.0002x±3.8947（R²=0.999）.The eDNA analysis of the water samples from six sites in the eastern sea area of Shenzhen showed that the estimated density of Exaiptasia diaphana was 12.9～26.2 ind.·m^-3,and it was preliminarily believed that the density of Exaiptasia diaphana might be related to the content of phosphorus in the environment.This study shows that the eDNA technology can be used for qualitative and quantitative analysis of Exaiptasia diaphana in Shenzhen,with the advantages of high specificity,low detection limit and quantitative limit.In order to improve the efficiency of the determination of total phosphorus（TP）in the aquaculture tail water discharged into the sea.In this study,Microplate Reader was used instead of spectrophotometer to determine TP in aquaculture tailwater,and the digestion method of TP,the type of microwell plate and the amount of sample were optimized.The results show that there was no significant difference（P>0.05）between60 min of digestion using a temperature-controlled heating plate and 30 min of digestion using an autoclave;the sensitivity of the assay was better than other experimental groups（200μL for 96-well plate,200μL for 96-well quartz plate,200μL and 500μL for 48-well plate,500μL and 1 mL for 24-well plate）when using a 48-well plate with 1 mL of sample.The linear correlation between phosphate concentration and absorbance was best（R²=0.9999）for this group and the 96-well plate spiked with 200μL;the accuracy and precision validation of TP determination in culture tailwater showed that the relative error（RE）and relative standard deviation（RSD）of the 48-well plate spiked with 1 mL were higher than those of the other experimental groups（96-well plate 200μL,96-well quartz plate 200μL,48-well plate 200μL and 500μL,24-well plate 500μL and 1 mL）.The recoveries of disodium glycerophosphate（β-GLP）and sodium hexametaphosphate（SHMP）were 98.2%～99.6%and 93.4%～97.1%,respectively,and the limit of quantification（LOQ.）of TP were 0.25μmol·L^-1;the RE and RSD of 96-well ELISA plate spiked with 200μL were-14.03%～0.21%and2.63%～14.23%,respectively,and the recoveries forβ-GLP and SHM were 94.7%～99.0%and 88.9%～97.3%,respectively,and the LOQ of TP was 0.55μmol·L^-1.At TP concentrations of 0.55μmol·L^-1～6.4μmol·L^-1,the accuracy and precision of the two optimized methods meet the determination requirements and could be used for the determination of TP in culture tailwater.The 96 h-LC₅₀ of phosphate to Exaiptasia diaphana is 3.19 mmol·L^-1.Three phosphate concentration groups of natural seawater（treatment group A）,0.32 mmol·L^-1（treatment group B）and 2.23 mmol·L^-1（treatment group C）were set up to observe the Apparent morphology of Exaiptasia diaphana in different concentrations,and determine the density of zooxanthellae,the content of chlorophyll a and malondialdehyde（MDA）and the activity of related enzymes.The study found that compared with treatment A,treatment B increased the content of chlorophyll-a in the tissues,and there was no significant difference in the density of Chlamydomonas in the two tissues,but the content of chlorophyll-a in a single Chlamydomonas increased significantly.Treatment group C resulted in the discharge of symbiotic chlorella in the body of Exaiptasia diaphana,increased secretion of mucus,significantly decreased the content of chlorophyll-a in the tissue and the density of chlorella,and damaged the tissue of Exaiptasia diaphana,resulting in the increase of MDA content.Alkaline phosphatase（AKP）activity in treatment B and C decreased significantly.In the determination of superoxide dismutase（SOD）and catalase（CAT）activity,there was no significant difference between treatment B and treatment A at each time point,while in the control group,both SOD and CAT had peaks,and there was phosphate stress.The results show that there may be phosphorus limitation in the sea area.Proper addition of phosphate will promote the content of chlorophyll-a in the tissue of Exaiptasia diaphana,enhance photosynthesis,and speed up growth and reproduction,which may be the reason for the high estimated density of Exaiptasia diaphana in Shatanhai Bay,Shenzhen.