Preliminary Study On The Epidemiology Of Hepatopancreatic Necrosis Syndrome In River Crab Fry

Eriocheir sinensis,also known as river crab(hereinafter referred to as river crab),is an important aquaculture species.In 2015.a new type of river crab disease,hepatopancreatic necrosis syndrome(HPNS),broke out in Jiangsu Province.which caused serious economic and restricted the development of river crab breeding industry to a certain extent.At present,it has been clear that HPNS occurs in adult crabs,and the related pathological and etiological studies have been partly reported.However,the specific pathological changes and etiology of HPNS are not clear,even it is impossible to carry out study on targeted prevention and control measures.To summarize,the research on HPNS of river crab is still in the primary stage,and most of which focus on adult crabs,whether the occurrence of HPNS in seedlings has rarely been reported,not to mention the etiology of HPNS.In order to understand the incidence of HPNS in crab seedlings and explore its etiology and pathogenesis preliminarily,epidemiological investigation,pathological observation and microbiology analysis were carried out in this paper.This paper is divided into the following three parts:1.Epidemiological investigation of HPNS in crab seedlingsIn order to understand the incidence of HPNS in river crab seedlings,an epidemiological investigation of HPNS in river crab seedlings in Jiangsu Province was carried out.The results showed that HPNS appeared in larval crabs of crab seedlings from 2017 to 2018.but not in megalopa and juvenile crabs.At the time of onset,the scale of the disease onset was small,the morbidity and mortality were low and other crustaceans in the same pond had no HPNS symptoms;the main time of the disease onset was from May to July:it only occurred in individual ponds in Xinghua and Danyang areas of Jiangsu Province.The diseased crabs with HPNS were characterized by decreased vitality,appetite,motility impaire and decreased hypoxia tolerance.Anatomical examination revealed albinism even atrophy of hepatopancreas.blackening of gill tissue and massive hydrocele in abdominal cavity of HPNS diseased crabs.Preliminary results of screening for pathogenic factors showed that the physical and chemical factors of water quality such as temperature.pH,ammonia nitrogen content and hydrogen sulfide content were within the tolerance range of normal growth of river crabs in the onset area.In terms of pathogen carrying in seedlings,no common crustacean pathogens such as white spot syndrome virus(WSSV),infectious hypodermal&haematopoietic necrosis virus(IHHNV),Spiroplasma eriocheiris,Hepatospora eriocheir were found in the megalopa;lower positive detection rate of IHHNV in juvenile crabs;higher positive detection rate of WSSV and IHHNV in larval crabs;no positive detection rate of S.eriocheiris and H.eriocheir in HPNS diseased crabs.However,the positive detection rates of WSSV and IHHNV were 86.67%and 26.67%respectively in larval crabs from HPNS onset pools of some batches.Especially,the HPNS diseased crabs carried both IHHNV and WSSV.In terms of the amount and composition of culturable bacteria,there was no significant difference between the amount of culturable bacteria isolated from hepatopancreas of HPNS diseased crabs and that of healthy crabs,but the dominant bacteria changed.Aeromonas and Pseudomonas became dominant bacteria.The above results showed that HPNS occurred in larval crabs of crab seedlings,but the incidence of HPNS was small.the morbidity and mortality were low,and the occurrence of HPNS was species-specific.It is preliminarily considered that the occurrence of HPNS in crab seedlings is not significantly related to physical and chemical factors of the water,but may be related to pathogens carried in crab seedlings.2.Pathological study of HPNS diseased seedlingsIn order to make a preliminary exploration of the etiology of HPNS in seedlings,the hepatopancreas of HPNS diseased seedlings were observed by histopathology and cytopathology.The results showed that vacuolar degeneration of hepatopancreatic tubules.decrease of secretory cells(B cells)and absorptive cells(R cells).basement membrane rupture and massive necrosis and exfoliation of epithelial cells were observed under optical microscope.Under electronic microscope,the hepatocytes of diseased crabs showed severe vacuolar degeneration.nuclear deformation.chromatin edge collection.nuclear membrane lysis.mitochondrial cristae reduction or disappearance.holistic vacuolation.and lysosome content increased significantly.The results showed that the hepatopancreas of HPNS diseased crabs had obvious pathological changes and their normal physiological functions were weakened,pathological changes are common in other aquatic animals infected by viruses.It is preliminary deduced that the occurrence of HPNS in crab seedlings may be related to the pathogen carried by crab seedlings.3.Preliminary study on microflora of hepatopancreas of HPNS in river crab seedlingsIn order to further explore the etiology and pathogenesis of HPNS in crab seedlings,the structure of microflora of hepatopancreas in the following five groups was studied by denaturing gradient gel electrophoresis(PCR-DGGE):healthy crabs.HPNS diseased crabs,crabs which onlv carry WSSV.crabs which only carry 1HHNV and crabs which carry both IHHNV and WSSV.The results showed that,in terms of the diversity of hepatopancreas microflora,the diversity of hepatopancreas mcroflora in HPNS group was lower than that in healthy group.In terms of relative abundance of some genera,compared with healthy group,the relative abundance of' Aeromonas.Pseudomonas and other pathogenic bacteria in hepatopancreas microllora of HPNS group and other three groups which carried pathogen increased,the abundance of beneficial bacteria,such as Sphingomonas and Sphingobacterium,which are related to the color of hepatopancreas and contain carotenoids,decreased.In terms of the similarity of bacterial microflora,the HPNS group and the group which carried both IHHNV and WSSV were clustered first(similarity 0.72).then with the group which only carried IHHNV(similarity 0.69)and the group which only carried WSSV(similarity 0.62).and finallv with the healthy group(similarity 0.56).indicating that the structure of hepatopancreas microflora in HPNS group was similar to that of the group which carried both IHHNV and WSSV,and the difference was significant between HPNS group and healthy group.The results showed that the decrease of the diversity of hepatopancreas microflora and the change of relative abundance of hepatopancreas microflora caused the decrease of the physiological function of hepatopancreas.and then HPNS appeared.It was preliminarily speculated that the cause of the change ofthe microllora might be related to carrying pathogens.especially carrying both IHHNV and WSSV.In addition.Braclvrhizohium was not found in the HPNS group.while was dominant bacteria in the other four groups.indicating that the HPNS diseased crabs had stopped feeding for a long time,and the other four groups' crabs were still feeing normally.It was preliminarily believed that under the aquaculture conditions.the physiological function of hepatopancreas declined due to larval crabs carried both IHHNV and WSSV.then crabs stopped feeding and gradually developed into HPNS.Based on the above results,it can be concluded that HPNS occurred in larval crabs of river crab seedlings,but the incidence of HPNS was small,the morbidity and mortality were low,and the occurrence of HPNS was species specificity.It is preliminarily deduced that the occurrence of HPNS in crab seedlings may be related to the carrying both IHHNV and WSSV.and the carrying both IHHNV and WSSV may lead to the decline of normal physiological function of hepatopancreas.making crabs stop feeding,and then gradually develop into HPNS.