Comparative Transcriptome Analysis Of Swimbladder And Expression Of Chemokinome Superfamily In Catfish(Ictalurus Spp.)Provide Insights To The Hypoxia Tolerance

Oxygen is indispensable for all aquatic aerobic organisms.Especially for the aquaculture industry with high stocking density,hypoxia often leads to high levels of mortalities of aquaculture species.If not,hypoxia can lead to suppressed growth,and depression of the fish immune system,causing increased susceptibility to diseases,which taken together brings about huge economic losses to aquaculture industry.Catfish(Ictalurus spp.),as a member of Siluriformes,is not only the major aquaculture species in the United States,but also widely cultured in China.Compared with many other cultured fishes,catfish is more tolerant to low oxygen stress,making it one of the best models to understand the mechanisms of hypoxia responses.Hence,the study on the mechanisms of catfish response to hypoxia is of great significance for providing a theoretical basis for the breeding of aquaculture species,and enhancing the aquaculture industry efficiency.Swimbladder is a gas-filled organ in most teleosts,contributing to regulate fish buoyancy.It has been hypothesized as a homolog of the mammalian lung,but the molecular evidence is scarce.Besides,many potential functions of swimbladder have been neglecting.The catfish transcriptome has been well studied from various tissues,left the vacancy in swimbladder transcriptome.Catfish is a good model to explore the shape of swimbladder morphology as catfish displays a diversity in swimbladder shape and number of chambers.The goals of this thesis are to conduct comparative transcriptome analysis to identity enriched genes and DEGs among swimbladders of three catfish species,and conduct transcriptome analysis in catfish swimbladder to provide insights to the responses to hypoxia through examing gene expression signatures,and for the genomic characteristics and expression profiles of the representative DEGs(chemokine)to account for their participations in responses to hypoxia stresses and bacterial infections.The main results were listed as follows:(1)In order to examine gene expression patterns in catfish swimbladder and DEGs among swimbladders of three catfish species,we conducted transcriptomic comparisons of adult and fingerling swimbladders in channel catfish,blue catfish and hybrid catfish utilizing high-throughput RNA-Seq technology.A total of 1159.2 million 100bp paired-end raw reads were generated and 1.04 ×1010 clean reads were used for subsequent analysis after trimming and normalization.A total of 4,612 DEGs with FDR<0.05 and absolute value of fold change>2 were identified through pairwise comparisons among channel,blue and hybrid catfish at the same stage.Among which,there were 3,346 and 1,639 DEGs found from fingerlings and adults between the comparison of channel and blue catfish,respectively.There were 603 and 338 DEGs found from fingerlings and adults between the comparison of channel and hybrid catfish,respectively.While there were 239 and 270 DEGs indentified from fingerlings and adults between the comparison of blue and hybrid catfish,respectively.Based on the annotated results of GO and KEGG,a total of 2,362 DEGs were identified to be involved in Wnt signaling pathway,Hedgehog signaling pathway,cell adhesion molecules(CAMs),focal adhesion,ECM-receptor interaction and neuroactive ligand-receptor interaction.The candidate DEGs and pathways identified in this research set the foundation for future studies on morphology and functions of swimbladder in catfish as well as other teleosts.(2)In order to reveal gene expression patterns and pathways involved in response to hypoxia in channel catfish,RNA sequencing was conducted using swimbladder samples of fingerling and adult channel catfish under low oxygen and normal conditions.A total of 529 million 100bp paired-end reads were generated and 4.69 X 109 clean reads were used for subsequent analysis after trimming and normalization.PCA analysis reflected the more significant differences between control and hypoxia treatment group in fingerlings than in adults.A total of 3,053 DEGs were identified after hypoxia stress in fingerlings,with 2,397 DEGs up-regulated and 656 DEGs down-regulated.In contrast,a total of 477 DEGs were found in adults,with 288 up-regulated and 189 down-regulated.Enrichment and pathway analysis of the differentially expressed genes showed the importance of the chemokine signaling pathway,HIF-1 signaling pathway,mTOR signaling pathway,MAPK signaling pathway,PI3K-Akt signaling pathway cell adhesion molecules(CAMs),focal adhesion,ECM-receptor interaction,neuroactive ligand-receptor interaction,and Hedgehog signaling pathway.The candidate DEGs and pathways identified in this study set the foundations for future studies on hypoxia respones in catfish as well as other teleost species.(3)A complete set of 17 CXC chemokine ligand(CXCL)genes was systematically identified and characterized from channel catfish genome.Phylogenetic analysis allowed identifications of the 17 catfish CXC chemokines.Extensive comparative genomic analyses supported their annotations and orthologies,revealing the existence of fish-specific CXC chemokines and the expansion of CXC chemokines in the teleost genomes.The analysis of gene expression after bacterial infection indicated the CXC chemokines were expressed in a gene-specific.CXCL 11.3 and CXCL20.3 were expressed significantly higher in resistant fish than in susceptible fish after ESC infection,while CXCL20.2 were expressed significantly higher in resistant fish than in susceptible fish after columnaris infection.The expression of those CXC chemokines,therefore can be a useful indicator of disease resistance.A similar pattern of expression was observed between resistant and susceptible fish with biotic and abiotic stresses,ESC,columnaris and hypoxia,suggesting that high levels of expression of the majority of CXC chemokines,with exception of CXCL11,CXCL 12 and CXCL20,are related to disease susceptibility or hypoxia intolerance.(4)A complete set of 64 CC chemokine ligand(CCL)genes was systematically characterized from the channel catfish genome through thoroughly data mining of existing genomic resources.Extensive phylogenetic and comparative genomic analyses supported their annotations,allowing establishment of their orthologies,revealing fish-specific CC chemokines and the expansion of CC chemokines in the teleost genomes through lineage-specific tandem duplications.With 64 genes,the channel catfish genome harbors the largest numbers of CC chemokines among all the genomes characterized to date,however,they fall into 11 distinct CC chemokine groups.Analysis of gene expression after bacterial infections indicated that the CC chemokines were regulated in a gene-specific and time-dependent manner.While only one member of CCL19(CCL19a.1)was significantly up-regulated after Edwardsiella ictaluri infection,all CCL19 members(CCL19a.1,CCL19a.2 and CCL19b)were significantly induced after Flavobacterium columnare infection.In addition,CCL19a.1,CCL19a.2 and CCL19b were also drastically up-regulated in ESC-susceptible fish,but not in resistant fish,suggesting potential significant roles of CCL19 in catfish immune responses.High expression levels of certain CC appeared to be correlated with susceptibility to diseases and intolerance to hypoxia.(5)A set of 30 CC chemokine recptor(CCR)genes and 8 CXC chemokine receptor(CXCR)genes were identified and annotated from the channel catfish genome.Extensive phylogenetic and comparative genomic analyses were conducted to annotate these genes,revealing fish-specific CC chemokine receptors,and lineage-specific tandem duplications of chemokine receptors in the teleost genomes.With 30 genes,the channel catfish genome harbors the largest numbers of CC chemokine receptors among all the genomes characterized.Analysis of gene expression after bacterial infections indicated that the chemokine receptors were regulated in a gene-specific manner.Most differentially expressed chemokine receptors were up-regulated after ESC and columnaris infection.Among which,CXCR3 and CXCR4 were observed to participate in immune responses to both Edwardsiella ictaluri infection and Flavobacterium columnare infection,indicating their potential roles in catfish immune activities.In addition,CXCR3.2 was significantly up-regulated in ESC-susceptible fish,and CXCR4b was mildly induced in ESC-resistant fish,further supporting the significant roles of CXCR3 and CXCR4 in catfish immune responses.CXCR4b and CCR9a were both up-regulated not only after bacterial infection,but also after hypoxia stress,provide the linkage between bacterial infection and low oxygen stresses.These results should be valuable for comparative immunological studies and provide insights into their roles in disease and hypoxia stress responses.