| Turbot(Scophthalmus maximus L.)is one of the main cultured species of flounders.It is cultivated in more than a dozen countries in Europe,the Americas and Asia.Due to its rapid growth,high nutritional value,and strong resistance to stress,turbot was introduced to China from the UK in the 1990 s by Academician Lei Jilin of the Yellow Sea Fisheries Research Institute of the Chinese Academy of Fishery Sciences,initiated the “Green house + deep well sea water” of the flow-through factory farming model.Turbot,after conquering many technical difficulties in industrial breeding,such as difficult oviposition,high rate of bleaching,and low survival rate,will soon be promoted in northern coastal areas,and it will be an important economy in coastal areas in the north of China.Large-scale breeding of farmed species has produced tremendous economic and social benefits,and has become a pillar industry for marine aquaculture in China.In order to ensure healthy,stable,and sustainable development of the turbot aquaculture industry,a certain number of turbot families are dedicated to the study of the genetic improvement of turbot traits every year.After more than 10 years of turboprop breeding,this group has ample experimental fish material and has accumulated rich experience in breeding.Based on years of breeding experience and understanding of the status of the turbot industry,the laboratory team learned that the bacterial disease in the turbot breeding industry has severely constrained the cultivation of turbot turtles and the cultivation of adult fishes.The content of this study is divided into two parts.The first part is the1-month-old turbot seedlings and their associated experimental materials(such as bait,aquaculture water,etc.)for the spontaneous outbreak of ascites disease when constructing the turbot family in Haiyang Base of this laboratory.)Fluorescencequantification and high-throughput analysis were performed.The second part of the study is to evaluate the genetic parameters of disease resistance character and bacterial quantitative analysis of 6-month-old turbot.The research results are as follows:1.Ascites in turbot is a bacterial disease in which multiple pathogens can independently cause disease.To explore the causes of outbreak of ascites in 1 month turbot of fluid factory farming mode,experiments were conducted to screen various pathogenic bacteria of turbot,ejaculated sperm cells,ovum,biological bait and aquaculture water in farms.Firstly,preliminary qualitative detection of Edwardsiella tarda,Vibrio anguillarum,Vibrio harveyi,Vibrio splendidus,and Vibrio alginolyticus was performed on the ascites of juvenile fish using fluorescent quantitative real-time PCR technique,supplemented by sequencing.The results showed that Vibrio anguillarum causing disease existed in the juvenile turbot.Then,the quantitative detection of bacteria in the biological bait and aquaculture water was carried out using the constructed TaqMan fluorescence quantitative PCR technology.The results showed that the rotifer,chlorella,Chlamydomonas and natural seawater were positive,and the copy numbers of Vibrio anguillarum were 0.216 copies/ng DNA,0.436copies/ng DNA,0.563 copies/ng DNA and 4.925 copies/ng DNA.2.The experiment explored the impact of environmental factors such as bio-feed and aquaculture water on the biodiversity of juvenile turbot and the relationship of the occurrence of ascites.The results showed that the number of OTUs(777)of ascites turbot was significantly higher than the number of healthy turbot OTUs(87),and the number of OTUs shared with biofeeds accounted for the total number of OTUs of two healthy juveniles respectively.91.95% and 81.85%,respectively,and the number of OTUs shared with natural seawater accounted for 26.44% and 23.93% of the total number of OTUs of juveniles in the two healthy states,respectively.It was indicated that the juvenile turbot larvae dominated the microbial composition of juvenile turbot larvae during the feeding of biofeed,and was related to the occurrence of ascites.According to the OTU commentary,the rotifers and chlorella in the diet contained a certain amount of Vibrio genus.Two sick fish detected Vibrio,and the vibrio genus bacteria accounted for 0.056% and 0.14% of the total bacterial count,respectively.The phylogenetic tree-heat map analysis of the first 100 OTUs with higher abundance revealed that Proteus mirabilis,Shigella spp.,and Enterobacter spp.are widely foundin juvenile fish,belonging to the same family of enterobacteriaceae as Edwardsiella tarda.However,there are currently no studies related to the pathogenicity of aquatic animals.The results of the cluster analysis combined the healthy juveniles with the food in the Golden Bay Artemia(Bohai Sea),Tibetan Artemia and Chlamydomonas,and the juvenile Artemia larvae(in Wudi),rotifers,and chlorella are clustered together,so it has been speculated that there is a correlation between larvae(Bohai Sea),rotifers,and chlorella in biological foods and outbreaks of ascites.3.TaqMan real-time quantitative PCR was used to quantitatively detect6-month-old turbot of Edwardsiella tarda infection.The bacterial load of the spleen,liver,intestine and rays of surviving turbot of 11 sampling sites such as 0h,12 h,24h,48 h,...,192 h,and 216 h was analyzed and compared.The results showed that in addition to spleen and liver,The gut and fins of the turbot were all positive,with an average load of 2.06±1.17copies/ng DNA and 1.32±0.49copies/ng DNA,respectively.With the progress of the experiment,Edwardsiella tarda proliferated massively in all tissues,especially in the intestine,indicating that the intestinal tract is the target organ for delayed infection by Edwardsiella tarda.The experiment also compared the bacterial load in turbot of the dead and surviving at multiple observation sites in the previous period.It was found that the bacteria load of surviving turbot at the same observation point was significantly higher than that of the dead turbot,indicating that the survival time can be used to breed a family or population of Edwards-resistant strains. |
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