Transcriptional Profiling In Amphioctopus Fangsiao Hemolymph After LPS And Poly I:C Stimulation And The Searching Of Key Immune Genes

Amphioctopus fangsiao is an economically important species in the coastal waters of China,with a delicious taste,richness in arginine,and high economic value.In recent years,due to the continuous increase of market demand and the increasing of offshore catch,the resources of wild A.fangsiao are gradually becoming exhausted.In order to protect the resources of wild A.fangsiao and meet the market demand,large-scale cultivation of A.fangsiao has become the focus of solving the problem.In invertebrates,innate immunity is the main mechanism that the body performs immune prevention and control.It plays the dual functions of cellular immunity and humoral immunity,which is important for recognizing and killing exogenous invading pathogens.That hemolymph cells can accurately recognize PAMPs(Pathogenassociated molecular patterns in pathogenic microorganisms),such as LPS(Lipopolysaccharide)and Poly I:C(Polyinosinic-polycytidylic acid)to initiate innate immune responses and produce immune responses can resist the invasion of pathogenic microorganisms.That the immune response mechanisms of A.fangsiao hemolymph stimulated by these two PAMPs can help us understand the innate immune response mechanisms of A.fangsiao and even cephalopods,which is of great value for the prevention and control of A.fangsiao aquaculture diseases.At present,transcriptome sequencing and analysis technology has been widely used in immunological research.That using this technology to analyze the RNA expression differences of hemolymph in A.fangsiao under the stimulation of different PAMPs is a convenient method to predict and reveal the functions of differential genes.This process can help identify the key genes and response mechanisms in the immune process.In this research,we conducted transcriptome analysis of hemolymph in A.fangsiao treated with LPS and Poly I:C,followed by WGCNA(weighted gene coexpression network analysis).The hemolymph were collected at 6h and 24 h after LPS stimulation,and 1,312 differentially expressed genes(DEGs)were identified.Similarly,the hemolymph were collected at 6h and 24 h after Poly I:C stimulation,and 1,369 differentially expressed genes(DEGs)were identified.Subsequently,we performed GO and KEGG functional enrichment analyses based on these genes.A large number of immune-related terms such as Innate immune response GO term,Immune system process GO term,JAK-STAT signaling pathway,Toll-like receptor signaling pathway,NOD-like receptor signaling pathway,TNF signaling pathway,chemokine signaling pathway,Leukocyte transendothelial migration,and NF-kappa B signaling pathway were found.Afterward,we constructed an immune-related protein interaction network based on genes from immune pathway and identified 28 hub immune genes including EGFR,JUN,MAP2K1,ACTG1 and other 24 hub genes after two stimulations.These hub genes are then analyzed and discussed in combination with signaling pathways.Based on the union of DEGs between the two stimulation modalities,we performed WGCNA using the FPKM value of 2,343 genes.We combined the immune stimulation time and expression model,which identified six modules with the strongest correlation.We performed functional enrichment analyses using genes from six modules and found a large number of immune-related terms,such as Cell surface receptor signaling pathway GO term,Innate immune response GO term,Immune system KEGG term,Bacterial infectious disease KEGG term.Moreover,we constructed a connectivity network based on the gene connectivity,and a protein–protein interaction network was constructed based on the protein–protein interaction relationship.Nineteen hub genes related to immunity including PKMYT1 and NAMPT were identified.We found that the processes related to inflammation,apoptosis,DNA repair and ubiquitination were the main hub genes stimulated by the two PAMPs.These reactions may play an important role in the immune response of A.fangsiao.In this study,a large number of immune terms and hub genes were discovered by transcriptome sequencing and analyzing of hemolymph in A.fangsiao after LPS and Poly I:C stimulation.These results will provide important guidance for the study of immune gene functions of A.fangsiao,laying a foundation for revealing the cellular immune defense and pathogen infection mechanism of cephalopods.Thses findings also provide ideas for disease prevention and control in A.fangsiao culture.