| "Zombie disease" of Exopalaemon carinicauda was a new epidemic that has appeared in pond breeding of E.carinicauda in coastal areas of Jiangsu Province since the winter of 2018,causing great economic losses to the local breeding industry of E.carinicauda.At present,there are no reports on etiology,molecular response mechanism,artificial prevention and control of "zombie disease".So,studying these issues can provide data and theoretical support for pathogenesis,artificial prevention and control of "zombie disease".In this paper,firstly,the epidemiological characteristics of "zombie disease" are deternined,and the pathogen is isolated and identified.Secondly,the whole genome information of the pathogen is analyzed.Thirdly,the molecular mechanism of Exopalaemon carinicauda in response to the pathogen infection is revealed.Finally,the effects of adding alginate oligosaccharides(AOS)on enhancing immunity and resistance to pathogen infection of E.carinicauda are explored.The main research contents are as follows:(1)Epidemiological characteristics,pathogen isolation and identification of "zombie disease" of E.carinicauda.In order to determine the epidemiological characteristics and pathogen of "zombie disease" of E.carinicauda,epidemiological investigation,pathological observation,pathogen isolation and identification have been used in this experiment.The results showed that from 2018 to 2021,the fastigium of the disease occurred from February to May each year,with the incidence rate of 5%-30% and the mortality rate of 3%-10%.The pathological change of hepatopancreas was the most serious,and a large number of colonizing bacteria existed in the gill and hepatopancreas tissues.A dominant strain MQ2101 was isolated from hemolymph of diseased shrimp.The symptoms of artificially infected shrimp were same to natural infected,and the same strain was also isolated from the artificially infected shrimp,which was in line with Koch’s rule.The strain MQ2101 was identified as Metschnikowia bicuspidata through morphological observation,physiological and biochemical identification,and comparative analysis of 18 S r RNA sequence.MQ2101 was highly pathogenic to E.carinicauda,with a lethal concentration of 50%(LD50)of 1.39×107 CFU/tail,and could survive and grow under the conditions of temperature 10-35 ℃,salinity 0-30 and p H 4-12.(2)The whole genome information of "Zombie disease" pathogen MQ2101 of E.carinicauda.In order to understand the genome information of Metschnikowia bicuspidata MQ2101,the whole genome sequencing and analysis were conducted on pathogen MQ2101 in this experiment.The results showed that the genome size was 15.98 Mb,containing 5 large fragments.The genome contained 3,934 predicted protein-coding genes,among which 3,899 genes had clear biological function annotation.1,055 virulence genes of MQ2101 were identified in PHI,and the number of genes with homology ≥50% was 275.235 virulence genes were identified in DFVF,among which 9 genes had homology > 90%.MQ2101 contained more carbohydrate active enzymes than the five closed relative yeasts.Two secondary metabolite gene clusters were predicted in MQ2101,one of which was a terpene metabolic gene cluster and the other was a gene cluster whose function was unknown.163 secreted proteins were predicted in MQ2101.Analysis of gene families between MQ2101 and five closed relative yeasts showed that strain MQ2101 contained 245 unique gene families,including unique 274 genes.(3)Proteomic analysis of hepatopancreas in E.carinicauda after MQ2101 infection.In order to explore the potential molecular mechanism of response to MQ2101 infection in E.carinicauda,proteomic analysis was performed on hepatopancreas of E.carinicauda(treated for 60 h)in saline control group and MQ2101 infection group.The results showed that a total of 5,078 proteins were identified,including 1,708 differentially expressed proteins(DEPs,1,311 up-regulated and 397 down-regulated).Further enrichment analysis of DEPs showed that the pathways of ribosome,lysosome,apoptosis and triglyceride metabolism were significantly enriched(p<0.05).Most of the DEPs in immune-related lysosome,apoptosis,autophagy,phagosome,endocytosis and NOD-like receptor signaling pathways were significantly up-regulated,suggesting that these immune-related metabolic pathways were significantly enhanced in hepatopancreas in response to MQ2101 infection.Most of the DEPs in metabolism-related triglyceride metabolic pathways were significantly up-regulated,while most of the DEPs in mucoglycans and other carbohydrate degradation pathways were significantly down-regulated,suggesting that after 60 h of MQ2101 infection,a large amount of stored sugars in shrimp may have been consumed to provide energy,and in order to continue to resist the pathogen,the shrimp began to metabolize lipid macromolecules to provide energy for the body reaction.In addition,some DEPs related to immunity and metabolism were verified by q RT-PCR,such as CTSB,CPs,DSP1,GPD,and ADH3,which may be potential determinants of the response of E.carinicauda to M.bicuspidate infection.(4)The effects of adding alginate oligosaccharides on enhancing immunity and resistance to pathogen infection of E.carinicauda.In order to explore the effect of adding AOS on immune enhancement of E.carinicauda and the feasibility of prevention and control of "zombie disease",the diets supplemented with 0(C),500(T1),1000(T2)and 2000 mg/kg(T3)of AOS were set up in this experiment.After feeding E.carinicauda for 60 days under the same conditions,correlation paremeters were detected.The results showed that the final body weight,total weight gain and specific growth rate in group T2 were significantly higher than group C(p<0.01),and the survival rate in group T3 was significantly higher than group C(p<0.01).There was no significant difference in ultrastructure among all groups.The activities of SOD and ACP in hepatopancreas and muscle of experimental groups were significantly higher than group C(p<0.05),the activity of AKP in hepatopancreas of group T2 was significantly higher than group C(p<0.05),and the activity of AKP in muscle of experimental groups was significantly higher than group C(p<0.05).The activity of PO in hepatopancreas of T2 and T3 groups,and in muscle of group T3 were significantly higher than group C(p<0.05).The relative expression level of SOD gene in hepatopancreas of experimental groups was significantly higher than group C(p<0.05),the relative expression level of pro PO gene in hepatopancreas of group T3 was significantly higher than group C(p<0.05),and the relative expression level of LGBP and CTL genes in hepatopancreas of experimental groups was significantly higher than group C(p<0.05).In MQ2101 challenge test,the survival rates of groups T1,T2 and T3 at 3-5days were significantly higher than group C(p<0.01),and the time of mortality reaching 50% was significantly later than group C.It was speculated that 1000 mg/kg AOS supplementation could improve the growth performance and immune capacity of Exopalaemon carinicauda with no side effects,and had certain prevention and control effect on Metschnikowia bicuspidate MQ2101.Based on the above research results,the following conclusions can be drawn:The pathogen of "zombie disease" of E.carinicauda is Metschnikowia bicuspidate MQ2101,which mainly infects hepatopancreas and gill tissues.The annual fastigium of the disease occurred from February to May,and the morbidity and mortality are5%-30% and 3%-10%,respectively.The genome size of MQ2101 is 15.98 Mb,containing 5 large fragments and 3,934 protein-coding genes,among which some virulence genes and secreted proteins may be directly involved in the pathogenicity of the strain.The specific genes contained in MQ2101 may be closely related to its own growth,reproduction and pathogenicity.After infection with MQ2101,E.carinicauda may respond to the pathogen infection process by enhancing a series of immune responses.After 60 h of infection,a lot of stored sugars may have been consumed,then,the shrimp began to metabolize lipid macromolecules to provide energy to continue to fight off pathogen.The suitable concentration of AOS supplementation can significantly improve the growth performance and immune capacity of E.carinicauda with no side effects,and has certain resistance to MQ2101. |
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