Effects Of Two Kinds Of Microalgae On Microbial Community Structure And Water Environmental Factors Of Larval Rearing Of Penaeus Monodon

Shrimp culture industry is the pillar industry of aquaculture in China,and high-quality seedlings are the key factors for the success of shrimp breeding.Although there have been many reports on the biological bait,water quality and bacteria related to the artificial breeding of shrimp in the past,due to the limitation of traditional research methods,the understanding of the breeding water environment,microbial diversity and its relationship with larval development and health is quite limited.This study adopted the research methods of shrimp breeding biology,phycology,hydrochemistry,immunophysiology,microbiology,molecular biology and other disciplines to study the growth and water quality factor changes of Thalassiosira weissflogii and Chaetoceros muelleri,as well as the effect of the two kinds of microalgae on the microbial community in the water environment,the intestinal microbial community in the larvae of Penaeus monodon and the development and metamorphosis of the larvae.The relationship between the water environment,the larvae and the microbial colony was understood from the molecular level,so as to provide scientific basis for the artificial breeding technology of Penaeus monodon.The main research results of this paper are as follows:1.The exponential growth periods of C.muelleri and T.weissflogii were 0-5 days and 0-3days respectively,and the maximum densities were 3×10⁶cells·m L^-1and 1.16×10⁶cells·m L^-1respectively.The number of bacteria in both microalgae decreased significantly during the exponential growth period.The number of vibrios in C.muelleri was almost zero on the 3rd to 6th day,while in T.weissflogii decreased to 0.13-0.29×10³cfu m L^-1on the 2nd to 4th day.The mass concentration of ammonia nitrogen and nitrite nitrogen were increased accompanied with the growth of microalgae,which was relatively high during the cultivation of T.weissflogii,reaching 0.06 mg·L^-1and 0.10 mg·L^-1,respectively,while in C.muelleri solution were 0.04 mg·L^-1and 0.04 mg·L^-1,respectively.In prawn seedling production,it is suggested that C.muelleri can be fed on the 4th to 6th day of culture,while T.weissflogii can be fed on the 3rd to 5th day of culture.2.T.weissflogii and C.muelleri can reduce the vibrios of seedling water,and the number of vibrios reached 0.1×10²cfu·m L^-1and 0.5×10²cfu·m L^-1in M₁stage,respectively.The total heterotrophic bacteria,ammonia nitrogen and nitrite nitrogen concentrations in the two groups showed a rising trend during the whole feeding larvae process,especially in the M₁-P₅period,and the total heterotrophic bacteria in the two groups reached about 3.0×10⁵cfu·m L^-1.The ammonia nitrogen and nitrite nitrogen concentrations in C.muelleri group were 0.08 mg·L^-1and 0.06 mg·L^-1,respectively,which were lower than those in T.weissflogii group(0.10mg·L^-1and 0.08 mg·L^-1).In P₅stage,the concentration of nitrite nitrogen in T.weissflogii group was significantly higher than that in C.muelleri group（p<0.05）.Digestive enzyme activities in all stages of larval development showed a trend of increasing first and then decreasing.The larval lipase activity reached the highest level in Z₁stage,and the amylase activity and pepsinase activity reached the highest values in M₁stage,furthermore,the lipase activity was the lowest and the pepsinase activity was the highest in larvae.The lipase activity and survival rate of the larvae fed with C.muelleri were significantly higher than those of T.weissflogii group in Z₁stage（p<0.05）.The survival rate of the larvae in P₅phase were 18%and 11%respectively in C.muelleri and T.weissflogii group.The larval protease activity was higher in the group fed with T.weissflogii,and the amylase activity and metamorphosis rate in larvae at mysid stage were significantly higher than those of C.muelleri group（p<0.05）.The alkaline phosphatase（AKP）and acid phosphatase（ACP）showed the same trend in all groups,i.e.they reached the highest level in the M₁stage and declined in post larval.Except for the mysid stage,the immune enzyme activity of larvae in C.muelleri group was higher than that of T.weissflogii group.3.Illumina Mi Seq sequencing platform was used to analyze the 16Sr DNA variable region of bacteria in the water,larvae and the algae liquid.The results showed that:The total number of OTUs in all samples was 13559.The OTU of intestinal bacteria from larvae showed a trend of increasing initially and then decreasing,and reached to the maximum in the Z₁stage.The diversity and richness of larval flora were the highest in Z₁stage,and the lowest in M₁stage and P₅stage,respectively.Moreover,the diversity of microflora in larvae fed with T.weissflogii was lower than that fed with C.muelleri.The bacteria community in the water and larval were composed with Proteobacteria,Bacteroidetes and Actinobacteria.Patescibacteria（52.54%）and Cyanobacteria（68.93%）was the dominant bacteria in the liquid of T.weissflogii and C.muelleri,respectively.Proteobacteria was dominant in larvae at Z₁stage,accounting for 85%and 82%respectively in C.muelleri and T.weissflogii groups.In M₁ stage,Actinobacteria（35.4%）accounted for a large proportion of larvae in C.muelleri groups,while the proportion of Bacteroides in T.weissflogii group was higher（52.64%）.At the family level,the bacteria community in the aquatic environment were mainly composed with Rhodobacteraceae and Flavobacteriaceae.C.muelleri liquid contained a large proportion of Virgulinella fragilis（68.8%）,while T.weissflogii liquid contained most unclassified bacteria and a small part of Microbacteriaceae（13.3%）.Microbacteriaceae（35.2%）and Saprospiraceae（47.4%）were the dominant bacteria in mysis of C.muelleri and T.weissflogii group,and Rhodobacteraceae accounted for a higher proportion in zoea.The microbial community structure in the fed algae liquid was quite different from that in the larval rearing water environment.In the process of feeding larvae,the bacterial community of the water and larval intestine might be affected temporarily by feeding conversion,but the intestinal bacteria from larvae is selective to the external bacteria and will not be affected by the dominant bacteria in the water.