Preliminary Studies On The Etiology And Pathogenic Mechanism Of Hepatopancreatic Necrosis Syndrome In Chinese Mitten Crab Eriocheir Sinensis

The Chinese mitten crab,Eriocheir sinensis,is one of the most economically important crustacean species in China,and its annual output value has been more than40 billion.With the development and improvement of culture model,the yield of Chinese mitten crab keeps rising year by year.However,with the flourish of crab culture industry and the increase of intensive rearing,frequent occurrence of disease has seriously affected the healthy development of crab culture.Jiangsu province is a big province of crab culture,and Xinghua city is awarded the title of"the first county of crab culture in China",and the crab culture industry has become the local pillar industry.However,the outbreak of hepatopancreatic necrosis syndrome in Chinese mitten crab in Xinghua city in 2015 has given a heavy blow to crab culture industry.Canhong typhoon passed through Xinghua city with serious damage in mid-July,hepatopancreatic necrosis syndrome occurred in more than 80%of the crab culture ponds,the morbidity was 30%-40%in most crab culture ponds,or as high as 90%-100%in some ponds,and caused huge economic losses to the farmers.In order to find out the etiology and pathogenic mechanism of hepatopancreatic necrosis syndrome in Chinese mitten crab in Xinghua city,so as to help farmers develop effective countermeasures,this paper was developed on the epidemiology,pathology,pathophysiology and transcriptomics and identified the relationship between hepatopancreatic necrosis syndrome and deltamethrin,pond cleaning medicine,which was much used in Xinghua city.The main results of this study were as follows:A systematic survey has been conducted to investigate the occurrence of hepatopancreatic necrosis syndrome in Chinese mitten crab in Xinghua city,in2015-2017,by collecting questionnaires,delivering training lectures,tracking crab culture in the whole process and providing a series of technology services.The results of the survey revealed that the main clinical symptoms of hepatopancreatic necrosis syndrome included decreased appetite,black thin and crisp shell,almost empty stomach and intestines,atrophic gills,weaken limbs,atrophic limb muscle,and the color of hepatopancreas changed from orange yellow to pale yellow and even white with atrophy necrosis.The characteristics of crabs with severe lesions were raised shells and water-filled abdominal cavities.With the outbreak of hepatopancreatic necrosis syndrome during the third and fourth moult of crabs in Xinghua city in 2015,this disease occurred in more than 80%of the crab culture ponds,the incidence was30%-40%in most crab culture ponds.The disease appeared in advance in 2016,it occurred when crabs moulted for the first time,and the incidence had reached 30%after the second moult.The incidence was relatively low in 2017,it was less than 5%.The most cases occurred during May and August,the water temperature was about between20 and 35 degrees,and the prevalence of this disease was highest when the water temperature was between 25 and 28 degrees.There was no significant difference in the incidence of male and female crabs,and there was no significant correlation between the incidence and the source of the offsprings.The incidence of hepatopancreatic necrosis syndrome of crab culture ponds,which used pyrethroids for pond cleaning,was significantly higher than that of these ponds used drugs with low toxicity,such as quicklime,bleaching powder and tea seed meal.The crab culture ponds with high incidence always had high water pH,no aerobic equipments,and too high coverage of aquatic plants.There was no effect regardless of the use of pesticides,antibacterial drugs or antiviral drugs in the early stage of this disease.The above findings suggested that hepatopancreatic necrosis syndrome was related to pyrethroids and low water quality.This study was also developed on the etiology by parasites examination,pathogenic bacteria isolation,challenge experiment and electron microscopy observation.In order to determine whether hepatopancreatic necrosis syndrome was contagious,diseased and healthy crabs were cohabited together and healthy crabs were fed with scrap diseased crabs.Results showed that no pathogenic microorganism was detected by etiology research,and the healthy crabs did not change whether they were fed with diseased crabs of cohabited with diseased crabs.Hepatopancreatic necrosis syndrome in Chinese mitten crab had no association with pathogens and was not contagious.The transmission electron microscope technique and routine paraffin-section technique were used to observe the different pathological tissues of the diseased crabs,with healthy crabs as control groups.Pathophysiological analysis of diseased crabs was conducted by biochemical analysis.Based on the main pathological characteristics and pathogenesis,it was suggested that this disease be nominated scientifically as hepatopancreatic necrosis syndrome.Microscopical observation results showed that pathological changes of different degrees appeared in gills,muscles and hepatopancreas of diseased crabs,the main pathological characteristics were vacuolization of the epithelial cells of hepatopancreas and transferred vacuoles appeared;With the severity of the conditions,hepatic cells were irregular-arranged,the number and volume of vacuoles and transferred vacuoles increased,and the number of granular materials in transferred vacuoles increased;When the disease was most severe,the hepatic tubles were so ruptured that substances in cells were leaked,the nucleus of hepatic cells were broken and hepatic cells appeared necrosis.Thickened gill tissue,enlarged gill cavity,increased number of hemocyte,and marginalized nucleus were the major pathological features of gills.The main lesion characteristics of muscles were relaxed muscular fibers and pyknotic nucleus.Ultramicroscopical observation results revealed that the main pathological characteristics were degenerative and necrotic hepatic cells,decreased number of lipid droplets,and swelled or fractured microvillus.The swelled mitochondria showed abnormal appearance,such as swell,disorganization and reduction or vanish of the crista.The endoplasmic reticula were dilated or fragmented into lamellar structure.As for gills,the chitin layer became thinner,the cell nucleus of epithelial cells were heterochromatinized,the finger shaped protuberance extended from the chitin layer was collapsed and disappeared,microvilli arranged disorderly and were fractured or vanished,the sub-cuticular space was expended,the number of mitochondria was decreased and mitochondria was swelled or vacuolar with the decrease of crista,the number of lysosome was increased and lysosome formed autophagosomes with vacuoles and vacuolar mitochondria.Bacteria particles appeared in the severely changed gill epithelium,but no inclusions and other virions.The main pathological changes in muscle cells included relaxed and cracked muscular fibers,melted sarcoplasmic reticulum,decreased number and volume of mitochondria,and pyknotic and marginalized nucleus.Pathophysiological analysis results showed that glucose content and GOT activity in hemolymph of diseased crabs were higher than that of healthy crabs,GPT activity was significantly higher than that of healthy crabs,whereas triglyceride content was significantly lower than healthy crabs.As for hepatopancreas,the liver glycogen content of diseased crabs was significantly lower than healthy crabs,ALP and ACP activities were lower than healthy crabs,and MDA content was significantly higher than healthy crabs.The above results showed that diseased crabs were threatened by oxidative stress and with low immunity.The structure of hepatopancreas,gills and muscles was damaged,and hepatopancreas had more severe lesions.Material metabolism was abnormal,especially carbohydrate and lipid metabolism.Transcriptomes of hepatopancreas of healthy and disease crabs were obtained by Illumina HiSeq2000 paired sequencing.DESeq software was used to screen the differentially expressed genes?DEGs?,and the selection standards were|log2Ratio|?1and q<0.05.Then the DEGs were annotated to homology sequence of NCBI,Uniprot,GO and KEGG database to obtain detailed descriptions.Compared with healthy crabs,1622 DEGs were detected in hepatopancreas of diseased crabs,644 genes were significantly upregulated and 978 genes downregulated.GO enrichment analysis showed that the expression of genes enriched in lipid metabolic process and carbohydrate catabolic process was downregulated.KEGG pathway analysis showed that the significantly enriched pathways were metabolism of xenobiotics by cytochrome P450,drug metabolism-cytochrome P450 and chemical carcinogenesis in addition to the material metabolism pathways,and the expression level of the DEGs enriched in the three pathways associated with drugs was all downregulated.In addition to the DEGs were related to the metabolism of sugar,fat and protein,the expression of the main drug-metabolizing enzymes was changed.The genes of cytochrome P450 family and glutathione S-transferase family was downregulated,however,the expression of carboxylesterase family genes was upregulated.Carboxylesterase is the major enzyme that breaks down pyrethroids,these results revealed that nutrient metabolism was abnormal,and the occurrence of the disease might have a certain relationship with pyrethroids.Deltamethrin is commonly used in Xinghua city in pond cleaning.In order to explore the relationship between deltamethrin and hepatopancreatic necrosis syndrome in Chinese mitten crab,an acute semi-static toxic test was carried out.The results showed that the LC₅₀ values of 24 h,48 h and 96 h were 4.29,3.48 and 1.32?g·L^–1,the safe concentration was 0.66?g·L^-1.Chronic toxicity test was conducted in different deltamethrin concentration including 96 h-LC₅₀/2,96 h-LC₅₀/5 and 96 h-LC₅₀/10,and the oxidative stress relative indexes,superoxide dismutase?SOD?activity,catalase?CAT?activity and malondialdehyde content?MDA?,were examined after being administrated with deltamethrin at intervals?6,12,24,48,72 h?.The results showed that although the crabs adjusted SOD and CAT activities to combat deltamethrin stress,the accumulation of MDA in the stress group was significantly higher than that in the control group during the whole experiment,which indicated that deltamethrin caused oxidative stress to the crabs.Histological structure and color of hepatopancreas were all changed observed on day 45 in different deltamethrin concentration,respectively.The color of heaptopancreas changed from orange yellow to light yellow,yellowish-white or pale-pink with necrotic erosion.Histopathological analysis showed that pathological changes of hepatopancreas,gills and muscles of crabs caused by deltamethrin stress were consistent with hepatopancreatic necrosis syndrome.Besides,we found that the categories,expression level and enriched pathways of DEGs of hepatopancreas under deltamethrin stimuli were also consistent with diseased crabs.Through the comparisons between the crabs under deltamethrin stress and diseased crabs on the clinical manifestations,pathological,physiological and transcriptional changes,we ultimately concluded that deltamethrin was an important cause of hepatopancreatic necrosis syndrome in Chinese mitten crab.To sum up,hepatopancreatic necrosis syndrome in Chinese mitten crab,commonly known as“Shuibiezi”disease by farmers,was caused by non living agents,and it was not contagious.Pathological changes in different degrees were observed in gills,muscles and hepatopancreas of diseased crabs,and the lesions of hepatopancreas were more serious no matter diagnosed based on clinical manifestations or pathological observation.Based on the main pathological characteristics and pathogenesis,it is suggested that this disease be nominated scientifically as hepatopancreatic necrosis syndrome.With deltamethrin stress,clinical manifestations,pathological,physiological and transcriptional changes of the crabs were consistent with diseased crabs,so we concluded that deltamethrin was an important cause of hepatopancreatic necrosis syndrome in Chinese mitten crab.In order to effectively prevent and control the disease,it was recommended that the farmers replaced pyrethroids with quicklime,bleaching powder and tea seed meal for pond cleaning.