Transcriptome Analysis Of IHHNV Infection In Juvenile And Adult Litopenaeus Vannamei And The Study Of LvCAP-3 Gene Function

Infectious hypodermal and haematopoietic necrosis virus(IHHNV)is the smallest known virus in shrimp,which causes runt-deformity syndrome(RDS)and leads to huge economic loss every year in penaeid shrimp farming.Studies have shown that the juvenile Litopenaeus vannamei(L.vannamei)is more susceptible to IHHNV infection than the adults.However,the specific pathogenesis of this susceptibility remains unclear.In order to investigate the mechanism of pathogenic differences in IHHNV infection of L.vannamei at different developmental stages,the juvenile and adult L.vannamei were studied by transcriptome high-throughput sequencing to analyze their response to IHHNV infection.Four immune genes closely related to antivirus were screened and their expression levels in different tissues were determined.The antiviral and immune function of caspase-3(CAP-3)gene was studied by RNA interference technology.The main contents and results are as follows:1.Transcriptome analysis of IHHNV infection in juvenile and adult L.vannamei:GO and KEGG enrichment were analyzed to search for differentially expressed genes(DEGs)related to immunity,growth and metabolism.The results showed that many immune-related genes of the juvenile and adult L.vannamei responded differently to IHHNV infection.For the adult L.vannamei,the expression of most immune-related genes was significantly up-regulated,which means that a cellular defense response was triggered after IHHNV infection.However,most immune-related genes in juvenile L.vannamei were inhibited,indicating that the immune system of juvenile L.vannamei is imperfect and makes it to be more susceptible to IHHNV.Similarly,the growth-related genes of L.vannamei were changed during IHHNV infection.For the juvenile L.vannamei,the growth-related genes were significantly down-regulated,which resulted in a growth hormone disorder and prevented the juvenile L.vannamei from growth.In the adult L.vannamei,most molting-related genes were significantly up-regulated,indicating that IHHNV infection leads the adult L.vannamei to early molting to eliminate pathogen.Metabolic process data showed that energy metabolism pathway was affected when L.vannamei infected with IHHNV.The adult L.vannamei infected with IHHNV can cause energetically costly and lead to the disturbance of the metabolism,activate complex immune systems to resist the invasion of pathogens.2.Screened and expression of immune candidate genes in L.vannamei: Based on the transcriptome data,four immune-related genes with significantly differential expression levels were screened in shrimp,which were perlucin-like protein(PLP),cathepsin C(Cath C),alpha-2-macroglobulin(α2M),and caspase-3(CAP-3).The expression of these four genes in different tissues(swimming appendages,muscle,gills,intestine,and hepatopancreas)were analyzed by q PCR.The results showed that the four genes were expressed in all detected tissues,but the expression levels were varied.The expression level of PLP was the highest in the hepatopancreas and lower in gills.CAP-3 was highly expressed in the hepatopancreas,followed by the gills,and the lowest in the swimming appendages.Cath C was highly expressed in the hepatopancreas and intestine.α2M was highly expressed in hepatopancreas and swimming appendages,and the lowest in muscle.3.The investigation on the impact of LvCAP-3 gene on IHHNV infection in L.vannamei: The IHHNV load of L.vannamei increased gradually within 24-120 h after injection of IHHNV,and the expression level of LvCAP-3 gene showed an upward trend.After LvCAP-3 gene was interfered,the IHHNV load of L.vannamei injected with IHHNV was higher compared to the control group,and the expression of certain immune and growth-related genes(HMC,CAT,ER and EGFR)was affected.The results showed that LvCAP-3 gene exhibits a certain degree of anti-IHHNV effect,and L.vannamei was more susceptible to IHHNV after LvCAP-3 gene was interfered.In conclusion,the study elucidated the mechanism of pathogenicity difference between juvenile and adult L.vannamei infected with IHHNV at the transcriptome level for the first time,and verified the function of the screened immune genes by RNA interference.The results of this study can help us understand the pathogenesis of IHHNV,explore the sensitive period of L.vannamei to IHHNV,and provide theoretical support for explore the immune defense mechanism of shrimp.