Effects Of Egg Yolk Antibody On Immunity And Disease Resistance Of Litopenaeus Vannamei And Research On Viral Nervous Necrosis Of Oplegnathus Punctatus

With the expansion of aquaculture scale,the increase of aquaculture density and the deterioration of aquaculture environment,the problem of aquaculture diseases is becoming more and more prominent.Diseases not only cause huge economic losses to aquaculture,but also seriously threaten the safety of water environment,the quality of aquatic products and human health.It has become the demand for healthy and green development of aquaculture to further study the internal causes of disease,epidemic law,infection and pathogenic mechanism,immune agents and host mechanism and other theoretical issues.This paper is divided into three parts.The effects of Vp _AHPND yolk antibody on growth,immunity and anti-Vp _AHPND infection of Litopenaeus vannamei,the effects of WSSV yolk antibody on anti-WSSV infection and immune indexes of Litopenaeus vannamei,and studies on viral nervous necrosis of spotted knifejaw.（1）.The effect of Ig Y on the growth,immunity and resistance to Vp AHPND infection of Litopenaeus vannamei.Different doses of Vp_AHPND Ig Y agent（0.2%and0.5%）were added in shrimp feeds and immunized the juvenile L.vannamei for 28 d.The growth rate and survival rate of the juveniles,the activity of immune enzymes and the relative expression level of immune genes in shrimp hepatopancreas were measured.The resistance to Vp _AHPND infection of immunized juveniles was determined by the immersion infection.The growth experiment results showed that after 28 d of immunization,there was no significant difference in the average growth rate,specific growth rate and survival rate of the juveniles between the immunized group and the control group.The immune function test results showed that after 14 d of immunization,compared with the control group,the 0.2%immunized group（0.2%Ig Y agent）gave significantly higher enzyme activities on phenoloxidase（PO）,superoxide dismutase（SOD）and lysozyme in the hepatopancreas.The relative expression level of antibacterial peptide（Crustin）gene was also significantly increased,while the relative expression level ofβ-1,3-glucan binding protein-lipoprotein（β-GBP-HDL）gene was significantly reduced in the 0.2%Ig Y agent group.The results of the immersion infection showed that the survival rate of the juveniles in the 0.2%Ig Y agent group was significantly higher than that of the control group,and the relative percentage of survival（RPS）of 0.2%Ig Y agent was 63.77%.In conclusion,oral administration of Vp _AHPND Ig Y agent will not adversely affect the growth and survival of L.vannamei.The 0.2%Ig Y agent can significantly enhance the immune function of L.vannamei by increasing the enzyme activities of PO,SOD,LZM and the expression level of Crustin gene,thereby improving the ability of shrimps to resist Vp _AHPND infection.This study implied that Vp _AHPND Ig Y agent has a strong application potential for the prevention of AHPND.（2）.The study on effect of Ig Y on resistance to WSSV infection of Litopenaeus vannamei.Different doses of WSSV Ig Y agent（0.2%and 0.5%）were added in shrimp feeds and immunized the juvenile L.vannamei for 28 d.The survival rate of the juveniles was measured within 14 days after artificial infection with WSSV.The activity of immune enzymes and the relative expression level of immune genes in shrimp hepatopancreas were measured after 3 d of WSSV infection.The results showed that compared with the control group without feeding Ig Y,the 0.2%Ig Y agent group gave significantly higher enzyme activities on superoxide dismutase（SOD）,phenoloxidase（PO）and significantly lower enzyme activities on acid phosphatase（ACP）,alkaline phosphatase（AKP）.The relative expression level of heat shock protein 70 gene（Hsp70）was also significantly increased,while the relative expression level of lectin gene（Lectin）andβ-1,3-glucan binding protein-lipoprotein gene（β-GBP-HDL）was significantly reduced in the 0.2%Ig Y agent group.The SOD activity was significantly increased and PO activity was not significantly different,while ACP and AKP activity were decreased in the 0.5%Ig Y agent group.The relative expression level of Hsp70 was also significantly increased and the relative expression level of Lectin was not significantly different,while the relative expression level ofβ-GBP-HDL was significantly reduced in the 0.5%Ig Y agent group.The artificial infection results showed that after 14 d of WSSV infection,the survival rates of shrimp in the 0.2%,0.5%Ig Y agent group and the control group were48.89%,87.78%and 0.00%,respectively.The survival rate of shrimp in the 0.2%and0.5%Ig Y agent group was significantly higher than that in the control group,and the survival rate of shrimp in the 0.5%Ig Y agent group was also significantly higher than that in the 0.2%Ig Y agent group.The specific Ig Y agent could change the course of disease,delay the onset and death of immune shrimp,and significantly improve the survival rate of shrimp over the same period.The results showed that oral specific Ig Y agent could affect the activity of immune enzymes and the expression level of immune genes in hepatopancreas of L.vannamei,significantly improve the ability of L.vannamei to resist WSSV infection.This study provides a basis for the application of Ig Y agent in the prevention and control of WSSV infection,and also provides a reference for the research of the anti-WSSV infection mechanism of Ig Y agent.（3）.The studies on viral nervous necrosis of Oplegnathus punctatus.Spotted knifejaw（Oplegnathus punctatus）,as a warm and tropical offshore fish,has the characteristics of delicate meat,high feed conversion rate and fast growth rate.In 2014,spotted knifejaw successfully achieved large-scale artificial breeding and breeding in China,and became a new marine aquaculture variety with potential development.Due to the short time of large-scale breeding,there are few diseases of spotted knifejaw.There was a large-scale death event occurred in several domestic breeding farms in March 2020.The cause of large-scale death of spotted knifejaw was viral encephalopathy and retinopathy,and the pathogen was red-spotted grouper nervous necrosis virus（RGNNV）,which was determated by epidemiological investigation,clinical symptoms observation,histopathological analysis（H&E）,transmission electron microscopy observation（TEM）,virus isolation and culture,tissue in situ hybridization（ISH）,virus sequencing and phylogenetic analysis.The results showed that the temperature was 24℃～26℃,salinity was 30,p H was 8 when the disease occurred The diseased larvae were mid-late larvae and juveniles with a length of 5 mm～13 mm.The infected larvae began to develop from10 days old at the earliest,and reached the peak at 25～26 days old.The cumulative mortality within 2～3 days was as high as 90%～100%.The diseased fish had weak swimming ability,low food intake or almost no food intake.Some diseased fish seedlings showed spiral swimming in the pool,and sometimes had a short period of frenzy and chaos.Histopathological analysis showed that the cells in the brain,retinal reticular layer and kernel layer of diseased fish seedlings were necrotic,and a wide range of vacuoles appeared.Transmission electron microscopy analysis showed that a large number of cells in the brain and retina of diseased fish were necrosis,cell membrane was broken and nucleus was swollen.A large number of virus particles with a diameter of about 25 nm are arranged in a lattice,or are dispersed in the cytoplasm as patches,or are combined with the endoplasmic reticulum.Some virus particles will form a“ring”structure in the middle of the biofilm.Inoculation of diseased fish tissue homogenate into perch brain cell line（LJB）showed obvious CPE.The virus was identified as RGNNV by RT-PCR amplification and sequencing.Tissue in situ hybridization analysis showed that both RNA1 and RNA2 of the virus were located in the lesion area,and the hybridization signals of the core layer and reticular layer of the retina and the brain tissue were strongly positive.RACE technology was used to determine the full-length RNA1 sequence of the virus,with the length of 3103nt.Based on the RNA1 and RNA2 gene sequences,the phylogenetic tree analysis was carried out on multiple virus strains from different seedling farms.The results showed that the gene sequences of virus strains from No.1 and No.2seedling farms were identical,and the homology with Dragon grouper nervous virus isolated from Xiamen was as high as 99.3%,suggesting that they were from the same infection source.There were some differences in the gene sequences between the virus strains from No.3 nursery and the virus strains from No.1 and No.2 nursery,suggesting that they came from different infection sources.This study confirmed that Sparus macrocephalus is a highly sensitive host of RGNNV,and RGNNV is a major threat to the breeding stage of spotted knifejaw.