Fish and Shellfish Immunology 91 (2019) 188–193 Contents lists available at ScienceDirect Fish and Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Full length article Post-transcriptional regulation through alternative splicing after infection with Flavobacterium columnare in channel catfish (Ictalurus punctatus) T Suxu Tana, Wenwen Wanga, Changxu Tiana, Donghong Niua,b, Tao Zhoua, Yujia Yanga, ∗ Dongya Gaoa, Zhanjiang Liuc, a The Fish Molecular Genetics and Biotechnology Laboratory, School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA b College of Life Sciences, Shanghai Ocean University, Shanghai, China c Department of Biology, College of Art and Sciences, Syracuse University, Syracuse, NY, 13244, USA ARTICLE INFO ABSTRACT Keywords: Columnaris disease has long been recognized as a serious problem worldwide which affects both wild and Fish cultured freshwater fish including the commercially important channel catfish (Ictalurus punctatus). The fun- Alternative splicing damental molecular mechanisms of the host immune response to the causative agent Flavobacterium columnare Bacterial infection remain unclear, though gene expression analysis after the bacterial infection has been conducted. Alternative Immune splicing, a post-transcriptional regulation process to modulate gene expression and increase the proteomic di- RNA-Seq versity, has not yet been studied in channel catfish following infection with F. columnare. In this study, genomic information and RNA-Seq datasets of channel catfish were used to characterize the changes of alternative splicing after the infection. Alternative splicing was shown to be induced by F. columnare infection, with 8.0% increase in alternative splicing event at early infection stage. Intriguingly, genes involved in RNA binding and RNA splicing themselves were significantly enriched in differentially alternatively spliced (DAS) gene sets after infection. This finding was consistent with our previous study in channel catfish following infection with Edwardsiella ictaluri. It was suggested to be a universal mechanism that genes involved in RNA binding and splicing were regulated to undergo differential alternative splicing after stresses in channel catfish. Moreover, many immune genes were observed to be differentially alternatively spliced after infection. Further studies need to be performed to get a deeper view of molecular regulation on alternative splicing after stresses, setting a foundation for developing catfish broodstocks with enhanced disease resistance. 1. Introduction loss of approximately $30 million annually [13]. In pond aquaculture system, mortality rates of adults and fingerlings can reach 60% and Columnaris disease, caused by Flavobacterium columnare, is the most 90%, respectively [14]. frequently occurring disease in the aquaculture industries around the The pathogenesis of columnaris disease has been extensively stu- world. First described by Davis in 1922 [1], the disease was named as died, while little is still known about the molecular mechanisms un- columnaris because the causal bacterial cells in a wet mount prepara- derlying the disease [15]. The adhesion ability, especially to gill tissues, tion of affected gills and fins were arranged in columnar aggregations. was determined to be a prerequisite for the successful colonization and The pathogen F. columnare is a Gram-negative, rod-shaped bacterium infection of F. columnare [4,16,17]. In channel cat fish, transcriptional that causes symptoms such as saddleback lesions, fin erosion, and gill analyses on gene expression after F. columnare infection were conducted necrosis [2,3]. It is pathogenic to a wide range of freshwater fish spe- in gill tissues, and expression levels of lectins and NF-κB signaling re- cies, including zebrafish (Danio rerio)[4,5], striped bass (Morone sax- lated genes were differentially regulated [18,19]. Genome-wide asso- atilis)[6], perch (Perca fluviatilis)[7], common carp (Cyprinus carpio) ciation study (GWAS) has been performed in backcross hybrid catfish, [8], Nile tilapia (Oreochromis niloticus)[9], rainbow trout (Oncor- and genes involved in phosphoinositide 3-kinase pathway were iden- hynchus mykiss)[10,11], and channel catfish (Ictalurus punctatus) tified to be significantly associated with columnaris disease resistance [4,5,12]. The channel catfish aquaculture industry in the United States [20]. In spite of extensive studies at the genomic and transcriptomic has been severely impacted by columnaris disease, causing economic levels, post-transcriptional regulation of the host responses to this ∗ Corresponding author. E-mail address: [email protected] (Z. Liu). https://doi.org/10.1016/j.fsi.2019.05.008 Received 21 January 2019; Received in revised form 29 April 2019; Accepted 4 May 2019 Available online 09 May 2019 1050-4648/ © 2019 Elsevier Ltd. All rights reserved. S. Tan, et al. Fish and Shellfish Immunology 91 (2019) 188–193 disease has not yet been known. samples, a RNA pool comprised of samples of all three time points was Alternative splicing is a prominent post-transcriptional regulatory formed together. mechanism for gene regulation in eukaryotes. It enhances the mRNA complexity by allowing the generation of multiple transcript variants 2.2. Identification of alternative splicing after infection from a single gene. The complexity may lead to altered mRNA locali- zation, stability, translation or decay, as well as proteomic diversity Alternative splicing profiles and their associated responding genes with different structures and functions [21,22]. Alternative splicing is following infection were determined by using the bioinformatic pipe- an extremely common regulatory mechanism [23]. For instance, nearly line as described in Tan et al. [41]. In brief, the paired-end raw data all multi-exon genes in human were estimated to undergo alternative underwent quality control process using FASTX toolkit [43] and splicing using high-throughput sequencing [24,25]. The inclusion of Trimmomatic [44]. Then, high-quality reads were aligned against the different exons spliced in mature mRNA is controlled by both specific channel catfish reference genome sequence with TopHat2 [45], and the cis-acting sequence elements and trans-acting regulatory proteins [26]. mapped reads were assembled to transcripts using Cufflinks 2.2.1 [46]. The differential use of splice sites results in complex splicing events ASTALAVISTA was utilized to identify and classify alternative splicing [27] and five basic types of alternative splicing events: 1) exon skip- events including five basic types and complex types [47]. Since the ping, 2) alternative 5′ splice site, 3) alternative 3’ splice site, 4) mu- number of alternatively spliced genes and the number of alternative tually exclusive exon, and 5) intron retention. splicing events were correlated with sequencing depth [48], the same Alternative splicing is a regulated process involved in many phy- number of high-quality reads was randomly selected for the subsequent siological processes, including defense responses [28–30]. It enhances identification and comparison of alternative splicing profiles between the level of diversity and flexible adaptation required for a functional control and infected samples. immune system [28]. One of the best examples is that alternative splicing facilitated the generation of diverse antigen receptors of lym- 2.3. Identification of differential alternative splicing (DAS) phocytes that can recognize a wide range of antigens [31]. Alternative splicing has been associated with disease resistance/susceptibility in The software rMATS [49] was used to define DAS by computing and various organisms ranging from plants to mammals [32–39]. For in- comparing the inclusion level [percent spliced in (PSI)] of certain al- stance, alternative splicing of Nmnat was reported to enhance neuro- ternative splicing event between two sets of RNA-Seq datasets. Taking protection under stress in Drosophila [33]; after infection of the mastitis the example of exon skipping, PSI is referred as the ratio of junction and virus, a novel spliced isoform of the complement component 4 gene was exon reads for the skipped exon to junction and exon reads for the exon increased in dairy cattle [36]. Alternative splicing is regarded as an and its two flanking exons [49,50]. An FDR-adjusted P value less than effective strategy for the regulation of immune responses against pa- 0.05 was used as criteria for DAS. DAS genes (genes with differential thogen infection in mammals and teleost fish [34]. alternative splicing events) were also determined. Alternative splicing was found to be important for host responses to stresses including heat stress [40] and enteric septicemia of catfish 2.4. Functional enrichment analysis (ESC) [41] in catfish. In an effort of understanding the involvement of post-transcriptional regulation through alternative splicing in catfish To reveal enriched functional classes in the DAS gene sets, a sta- after stresses, in this study, we used RNA-Seq datasets to determine the tistical overrepresentation test [51] was performed using the PANTHER alternative splicing profiles and functional associations after F. co- classification system (http://pantherdb.org). To use PANTHER, zebra- lumnare infection in channel catfish. fish orthologs were searched through BLAST program and ENSEMBL database (http://www.ensembl.org). 2. Materials and methods 3. Results 2.1. Data collection 3.1. Alternative splicing profiles after F. columnare infection The channel