Isolation And Drug Resistance Analysis Of Fusarium Solani And The Immune Response Of Litopenaeus Vannamei To Its Infection

Pacific white shrimp,Litopenaeus vannamei,is the shrimp with the largest production in the world.Up to now,the researches on the diseases of L.vannamei mainly focused on viral and bacterial infections.At present,the shrimp culture mode is mainly in extensive and high-density culture,which is easy to cause the deterioration of the culture environment and to provide conditions for the rapid spread of pathogens.There are few reports on the fungal infections.Also,the study of Fusarium disease in L.vannamei mostly focused on eggs or larvae rather than parent shrimp.Furthmore,the study on the immune mechanism of L.vannamei infected by Fusarium solani in the transcriptome level has not been reportedIn this study,a dominant strain of Fusarium solani was isolated from the overwintering parent of L.vannamei with black gill disease.Furthermore,the pathogenicity,drug resistance,and taxonomic status of this dominant strain were analyzed and identified.The RNA sequencing was first performed on L.vannamei haemocytes of the experimental group(F.solani infection,E group)and control group(Saline injection,C group).Immune-related genes with different expression trends were identified.Quantitative real-time PCR(qRT-PCR)were carried out to validate their expression.The results are shown as follows:(1)The pathogen causes the black gill disease of L.vannamei is F.solani.(2)Drug sensitivity analysis showed that the pathogen was sensitive to clotrimazole,Econazole,terbinafine,nystatin.(3)A number of 1,538,571,432 clean reads generated from RNA-seq were assembled into 19,970 genes from haemocytes of L.vannamei.3305 differentially expressed genes(DEGs),including 1980 up-regulated and 1325 down-regulated DEGs were obtained.Based on differential expression analysis,trend analysis,and functional annotation,several immune-related genes were identified for qRT-PCR analysis.Go enrichment analysis of DEGs showed that the DEGs were significantly enriched in protein phosphorylation,cytokinesis,protein kinase activity,transcription,and other processes.(4)FPKM analysis and real-time quantitative PCR verification of transcriptome showed that,compared with the control group,the expression of ALF and crustin increased rapidly in the early stage of infection(12h),which were the most rapid immune factors of shrimp to response to fungal infection.Clec and KRAS,PTPN11 in JAK/STAT pathway were significantly up-regulated at 24 h and 96 h after infection,indicating that JAK/STAT pathway was involved in the immune response of L.vannamei against F.solani.TLR1 and TLR2 showed different reactions to F.solani infection.Compared with the control group,The expression of TLR1 was significantly up-regulated in 24 h,while TLR2 was down-regulated in the process of infection.It is speculated that TLR1 may play a significant role in response to fungal infection.Cactus in the Toll pathway and Relish in the Imd pathway were significantly up-regulated at 24 h after fungal infection,indicating that both pathways were involved in the response of shrimp to fungal resistance.The expression level of Dscam was up-regulated at 48h and the highest expression level at 96h,which was the later immune factor for shrimp to resist fungal infection.The expression of SP,SP7,PPAE,and PO3 in the phenoloxidase activating system was significantly up-regulated in most phases.The first three may be involved in the process of activating proPO to PO3.In conclusion,F.solani was isolated from L.vannamei.Four kinds of drugs that can treat the disease were screened out by the drug sensitivity test.After infected by fungi,lectin and JAK/STAT pathway,Toll and Imd pathway,phenoloxidase system,and Dscam of shrimp are involved in the immune response.The results of this study laid a foundation for the study of the immune function of shrimp against fungi.