A Research On Microbial Diversity And Community Dynamics In Sea Cucumber Culture Ponds

Sea cucumber farming is one of the most important industrial aquaculturein Liaoning and Shandong coast of China.Sea cucumber farming has developed rapidly,in 2010 the national sea cucumber breeding area has reached 10000 hectares,and the output reached 10000 tons,making a commercial value of nearly 1 billion.However,compared with the rapid expansion of the farming area,the basic research related to farming technologyis still lagging behind.Microbes are producers,consumers and also decomposers in themarine ecosystem.They not only play important roles in the nutrient cycling and energy flow in the aquaculture ecosystems,but alsoplay substaintial rols to maintain a stable breeding environment.Meanwhile,there are close relationships between microbes and the breeded cultures.,Microbes often are disease-causing pathogens or condition causative pathogen that lead to disease of the breeded organisms.In the breeding process,the surplus bait produced by irrationally feeding,the manure produced by the feeded object and the abuse of the biological medicines,often lead severely damage to the water micro-ecological balance,causing water degradation and the death of the sea cucumbers.The health of the farming environment is closely related with the structure and function of microbial communities.Thus,studies on microbial community structure and changesare theoretically and practically significant for the aquaculture farming industy.In this study,we used a flow cytometry technich to study the planktonic microbial abundance in the marine aquaculture water;High-throughput sequencing were used to study the communities of fungi,bacteria and archea;A continuous flow analyzer was used to measure the environmental factors.The correlationship between microbes and environmental factors were deduced based on the data above;Using specific primers and the quantitative PCR technic,we managed to detect the gametes and spores of the bloomable algaes,to achieve the purpose of monitoring the outbreak of the moss.The goal of this study is to provide basic data for the ecological regulation model of marine microorganisms in marine culture ponds.Our main results are shown as follows:1 Distribution of planktonic microorganisms in sea cucumber culture pondsFlow cytometry was used to study the abundance of planktonic microorganisms and its relationship with the environmental factors.Results show:(1)Planktonic microorganisms detected Synechococcus,Picophytoplankton,Nanophytoplankton,ryptophyta,high and low acidcontents heterotrophic bacterioplankton,which the concentration of bacteria is very large,almost 106,but the amount of phytoplankton level is small,only 102-103.(2)Planktonic microorganisms generally began to grow in March,the peak period in June,in September has been in recession.(3)There is a certain correlation between planktonic microorganisms and dissolved oxygen,salinity,pH and total soluble nitrogen in the environment,and the influence of dissolved oxygen and pH on planktonic microorganisms is the largest.2 Analysis of microbial diversity in sea cucumber culture pondsThe DNA was extracted by water filtration membrane,and the high flux was measured,and the microbial diversity in the sea cucumber culture pond was studied by comparing the environmental factors.Results show:(1)Proteobacteria is an important component of bacteria,and Alphaproteobacteria is the main group.The results of CCA analysis showed that prokaryotes were controlled by pH,NO2--N,SiO2--Si,NH4--N,T.(2)Most of the dominant taxa in archaea are DHVEG-6.And most of the archaea such as DHVEG6,MHVG,GroupC3,MBGB,with all kinds of soluble nitrogen element has significant and even very significant correlation.The results of CCA analysis showed that the microbial community was controlled by Salinty and SiO2-Si.(3)Archaeplastida is the main component of eukaryotic microorganisms,while Chlorophyta is the most important component in Archaeplastida.CCA results showed that the biological groups were controlled by Salinty,T,pH,DO,NO2--N,N:P.3 Detection of spores and gametes of three species of algae in sea cucumber culture ponds by qPCRThe three mosses collected from the sea cucumber culture ponds were extracted by DNA and sequenced by 18S and ITS,and we designed specific PCR primers targeting the internal transcript region(ITS)of the ribosomal RNA genes(rDNA)of three macroalgal species.The specificity of these primers was verified in silico,in vitro and in situ.Results show:(1)Three strains of algae were compared by NCBI,are Ulva cf.linza(Enteromorpha linza)?Ulva sp.,Scytosiphon lomentaria.(2)Minimum detection limit of three algae:Ulva cf.linza is 1.66×10-4 ng/?L,200 copies of the ITS gene;Ulva sp.is 6.6×10-6 ng/?L,200 copies of the ITS gene;Scytosiphon lomentaria is 1.66×10-4 ng/?L,400 copies of the ITS gene.(3)In the water,there can only detect Scytosiphon lomentaria spores/gametes,and in 2014 December in water samples of scytosiphon spores/gametophyte copy number of ITS is about 1x 107 copies/mL in 2015,the highest in March,about 2.5 x 107 copies/mL;since June 2015 scytosiphon/spore gametophyte ITS copy number was reduced to 5x 106 copy the/mL,have since less than 4×106 copies/mL.