Study On The Eriocheir Sinensis Hepatopancreatic Lesion Syndrome And Relationship With Microbial Flora Diversity

Eriocheir sinensis hepatopancreatic lesion syndrome,also called "water scorpion" disease,usually occurs from spring to autumn.Outbreaks of this disease often occur at high temperature of summer.The disease can infect both young and adult crabs and cause mass mortalities of crabs,which has become an important factor restricting the healthy development of Chinese mitten crab breeding industry.In this study,the epidemiological investigation,pathogen detection,histopathological analysis and the diversity of microbial in tissue and culture environment of Chinese mitten crab in Jiangsu province were conducted.The study aimed to explore the prevalence of hepatopancreatic syndrome in crabs,potential pathogen species,pathological features,changes in microbial diversity after hepatopancreatic lesions of Chinese mitten crab,and provide a certain reference for the prevention and control of hepatopancreatic lesion syndrome of Chinese mitten crab.The main research contents are as follow:(1)Epidemiological investigation of hepatopancreatic syndrome of Chinese mitten crab.In order to analyze the occurrence characteristics and epidemic rule of hepatopancreatic syndrome of Chinese mitten crab in the breeding production in 2018,the targeted tracking samples were carried out in the main breeding area of crab in Jiangsu province,and the disease occurrence was recorded in detail.The results showed that the rate of hepatopancreatic syndrome in Chinese mitten crab was low in 2018 and only occurred in the sunmer.(2)Detection of potential pathogens of hepatopancreatic syndrome of Chinese mitten crab.The gills,hepatopancreatic and intestinal tissues of crabs were sampled for detection of White spot syndrome virus,hepatoenterosporidium,microsporidium and spiroplasma.Hepatopancreas from diseased crabs were aslo sampled for bacteria isolation and pathogenicity of the bacteria was investigated.The results showed that White spot syndrome virus were found both in diseased and healthy crabs,hepatoenterosporidium were found in healthy crabs,microspoiidiurm and spiroplasma were negative;The four potential pathogens of white spot syndrome virus,microsporidia,heparanas and spiroplasma may not be related to the hepatopancreatic lesion syndrome of Eriocheir sinensis;Pathogenicity test of Aeromonas hydrophila and Citrobacter freundii from diseased crabs showed that the two bacteria were pathogenic to crabs,and the LD50 of the two bacteria to crabs were 5.4×105 CFU/mL and 3.2×106 CFU/mL,respectively.(3)Histopathological study on hepatopancreatic syndrome of Chinese mitten crab.Histopathological analysis of various tissues from diseased crabs with hepatopancreatic syndrome was carried out.The results showed that histopathological analysis revealed that hepatopancreas,gill and muscles of diseased crabs exhibited obvious pathological changes,and hepatopancreas was the most severe lesions.The number of blood cells between the hepatic tubules of the diseased crabs increased,the basement membrane was ruptured,some hepatic tubules were edematous and degenerative,the cell structure was blurred,and the vacuoles become large.The gill leaf of diseased crabs was thickened,the epithelial cells were irregularly arranged,a large number of blood cells were filled and squeezed in the blood chamber,the keratinocytes were corrugated and arched,large vacuolation was occurred between the keratinocytes and epithelial cell,and there was a little amount of impurities between the gill leaves.The muscle fibers of the diseased crabs were loose,some muscle fibers were broken and the horizontal stripes were blurred.(4)Microbialflora diversity analysis of diseased crabs,healthy crabs,farming water and sediment.Gills,hepatopancreas,intestinal tissues of crabs,aquaculture water and sediment were sampled for high-throughput macro-genome sequencing analysis to analyze the microflora diversity of different tissues,farming water and sediment from diseased crabs culture area compared to these from healthy crabs.The results showed that there were significant differences in the composition of microflora at different classification levels.The dominant bacteria in various tissues of healthy Chinese mitten crab were Firmicutes,Tenericutes,Proteobacteria and Bacteroidetes;The dominant bacteriaflora in crabs with hepatopancreatic lesion syndrome is Proteobacteria,Firmicutes and Tenericutes.The dominant bacteria groups in farming water and sediment from healthy and diseased groups were Proteobacteria,Bacteroidetes,Actinobacteria,Firmicutes and Verrucomicrobia;Compared with healthy crabs,the gill tissues of diseased crabs had less Proteobacteria and Actinobacteria,had more Bacteroidetes;The contents of Proteobacteria in hepatopancreatic tissues decreased,Bacteroidetes and Firmicutes increased,and Chlorobacteria and Verrucomicrobia increased.The contents of Proteobacteria and Bacteroidetes increased in intestinal tissues,while those of Firmicutes decreased.The results provide a theoretical reference for further revealing the causes of hepatopancreatic lesion syndrome in Chinese mitten crab.