Vibrio Anguillarum Infection Responses In Octopus Ocellatus Larva And Targeting Of Key Immune Genes

Octopus ocellatus,affiliated to Octopodidae under the phylum Mollusca,is one of the most important economic species along the northern coastal areas of China.It has been considered as a candidate for aquaculture enhancement and release owing to its short life span and rapid growth.O.ocellatus is well received by consumers owing to its high nutrition,tender and delicious flavour.However,the aquaculture of O.ocellatus is hampered by a lot of problems,such as low survival rate,poor stress resistance,and instability of seed offering,for which the octopus sold on the market was mostly captured by the sea.Moreover,the farming O.ocellatus was susceptible to the infection caused by vibriosis.Vibrio anguillarum,which is a kind of conditional pathogen,is one of the most harmful pathogenic bacteria in fish and shellfish and could infect a variety of freshwater and marine animals all around the world.Therefore,in order to improve the current aquaculture model and the survival rate,and enhance its resistance to environmental stress of farming O.ocellatus,it is necessary to research its mechanism of immune response to foreign microorganisms.In this research,we identified the key immune-related genes and signaling pathways as a whole from the octopus larva infected by V.anguillarum to analysis their expression patterns and roles in immune response to bacteria.In this study,through the RNA-Seq analysis and the excavation of key immune-response genes,the immnue response mechanism of the species to V.anguillarum infection was better understood.And it can provide some reference for the immune response mechanism and key immune genes of shellfish and marine invertebrates to V.anguillarum infection.Blank control group,4h after infection experimental group and control group,12 h after infection experimental group and control group,as well as 24 h after infection experimental group and control group were carried out in this study,with three replicates in each group.The samples of octopus larva were stored in the liquid nitrogen.By RNA-Seq in total 12 samples from octopus larva,we got 625205152 reads,amongwhich there were 593464376 clean reads.As a non-model organism,there was no reference genome of O.ocellatus,so we split joint a reference genome with the transcriptome data,then compared the reads back to the linked reference genome for further statistical analysis.The data was further processed and analyzed for differential expression genes(DEGs),and the key immune-related genes,gene family,Go term and KEGG signaling pathway were targeted,verified and predicted.In this research,we use multiple approaches with RNA-Seq analysis,such as heatmap analysis of hierachical clustering,expression trend analysis,GO functional enrichment,KEGG signaling pathway enrichment,protein-protein interaction network analysis.A total of 724 DEGs identified in octopus larvae infected by V.anguillarum,we finally selected and identified 13 DEGs closely related to the immune process of this species,and 8 DEGs(ATG5?CALM1A?CREB3L1?DAB2IPB?GCLM?GANI1?PLCB3?PRKAR2AA)were deeply analyzed by RNA-Seq and qRT-PCR.We found 11 enriched immune-related KEGG signaling pathway significantly,in which several immune genes were involved in multiple signaling pathway.We speculated that these genes involved in multiple signaling pathway may play a bridge role in the immune response to the forgein microorganisms in O.ocellatus.We also made a deep analysis and discussion for the ATG family(autophagy-related gene family)?CALM1A gene(calmodulin 1a gene)?CREB3L1(Camp response element-binding protein 3-like 1)?GCLM gene(glutathione cysteine ligase modulatory subunit)?cGMP-PKG signaling pathway ? chemokine signaling pathway and Rap1 signaling pathway and made a prediction for their function in immune response in O.ocellatus.In this study,Venn diagram,heatmap analysis of hierachical clustering of DEGs,GO functional enrichment,KEGG signaling pathway enrichment,and protein-protein interaction network was firstly applied to the cephalopod immune response related RNA-Seq analysis,and several key immune-related genes in V.anguillarum infection responses were targeted and discovered in O.ocellatus,which provide rich gene resources in the following shellfish immunology research,and provide some references for the O.ocellatus and other shellfish aquaculture industury.