Columnaris causes severe mortalities among many different wild and cultured freshwater fish species, but understanding of host resistance is lacking. Catfish, the primary aquaculture species in the United States, serves as a great model for the analysis of host resistance against columnaris disease. Channel catfish in general is highly resistant to the disease while blue catfish is highly susceptible. F2 generation of hybrids can be produced where phenotypes and genotypes are segregating, providing a useful system for QTL analysis. To identify genes associated with columnaris resistance, we performed a genome-wide association study (GWAS) using the catfish 250 K SNP array with 340 backcross progenies derived from crossing female channel catfish (Ictalurus punctatus) with male F1 hybrid catfish (female channel catfish I. punctatus × male blue catfish I. furcatus).

Peptidoglycan recognition proteins (PGRPs) can recognize bacterial cell wall (peptidoglycan) and activate innate immune system. In addition to its function as pathogen recognition receptors (PRRs), PGRPs are also involved in directly killing bacteria, and regulating multiple signaling pathways. Recently, we have reported catfish PRRs including nucleotide-binding domain, leucine-rich repeat containing receptors (NLRs), retinoic acid inducible gene I (RIG-I) like receptors (RLRs), and Toll-like receptors (TLRs). In this study, we identified and characterized the PGRP gene family in channel catfish which included two members, PGLYRP-5 and PGLYRP-6. Phylogenetic analysis, syntenic analysis and protein structural analysis were conducted to determine their identities and evolutionary relationships. In order to gain insight into the roles of PGRPs in catfish innate immune responses, quantitative real-time PCR was used to investigate the expression profiles in catfish healthy tissues and after bacterial infection. Both PGLYRP-5 and PGLYRP-6 were ubiquitously expressed in all 12 healthy tissues, and most highly expressed in gill and spleen, respectively. Distinct expression patterns were observed for PGRPs after infection with Edwardsiella ictaluri and Flavobacterium columnare, both Gram-negative bacteria. After infection with E. ictaluri, both PGLYRP-5 and PGLYRP-6 were significantly down-regulated at a certain time-point, while both genes were generally up-regulated in the gill after infection with F. columnare. Collectively, these findings suggested that PGRPs may play complex roles in the host immune response to bacterial pathogens in catfish.

Along with the rapid advances of the nextgen sequencing technologies, more and more species are added to the list of organisms whose whole genomes are sequenced. However, the assembled draft genome of many organisms consists of numerous small contigs, due to the short length of the reads generated by nextgen sequencing platforms. In order to improve the assembly and bring the genome contigs together, more genome resources are needed. In this study, we developed a strategy to generate a valuable genome resource, physical map contig-specific sequences, which are randomly distributed genome sequences in each physical contig. Two-dimensional tagging method was used to create specific tags for 1,824 physical contigs, in which the cost was dramatically reduced. A total of 94,111,841 100-bp reads and 315,277 assembled contigs are identified containing physical map contig-specific tags. The physical map contig-specific sequences along with the currently available BAC end sequences were then used to anchor the catfish draft genome contigs. A total of 156,457 genome contigs (~79% of whole genome sequencing assembly) were anchored and grouped into 1,824 pools, in which 16,680 unique genes were annotated. The physical map contig-specific sequences are valuable resources to link physical map, genetic linkage map and draft whole genome sequences, consequently have the capability to improve the whole genome sequences assembly and scaffolding, and improve the genome-wide comparative analysis as well. The strategy developed in this study could also be adopted in other species whose whole genome assembly is still facing a challenge.

Nitric oxide is well known for its roles in immune responses. As such, its synthesizing enzymes have been extensively studied from various species including some teleost fish species. However, the NOS genes have not been characterized in channel catfish (Ictalurus punctatus). In this study, we identified and characterized three NOS genes including one NOS1 and two NOS2 genes in channel catfish. Comparing with the NOS genes from other fish species, the catfish NOS genes are highly conserved in their structural features. Phylogenetic and syntenic analyses allowed determination of NOS1 and NOS2 genes of channel catfish and their orthology relationships. Syntenic analysis, as well as the phylogenetic analysis, indicated that the two NOS2 genes of catfish were lineage-specific duplication. The NOS genes were broadly expressed in most tested tissues, with NOS1 being expressed at the highest levels in the brain, NOS2b1 highly expressed in the skin and gill, and NOS2b2 lowly expressed in most of the tested tissues. The most striking findings of this study was that the expression of the NOS genes are highly regulated after bacterial infection, with time-dependent expression patterns that parallel the migration of macrophages. After Edwardsiella ictaluri challenge, dramatically different responses among the three NOS genes were observed. NOS1 was only significantly in the skin early after infection, while NOS2b1 was rapidly upregulated in gill, but more up-regulated in trunk kidney with the progression of the disease, suggesting such differences in gene expression may be reflective of the migration of macrophages among various tissues of the infected fish. In contrast to NOS1 and NOS2b1, NOS2b2 was normally expressed at very low levels, but it is induced in the brain and liver while significantly down-regulated in most other tissues.

Toll-like receptors (TLRs) were the earliest characterized and the most extensively studied pathogen recognition receptors (PRRs). The majority of tetrapod TLR orthologs have been found in teleost fish. In addition, a group of "fish-specific" TLRs have been identified. In catfish, a number of TLR-related sequences have been reported, but systematic phylogenetic analyses have not been conducted. In this study, we conducted phylogenetic and comparative analysis of 20 catfish TLR genes against their counterparts from various species. TLR25 and TLR26 are TLRs identified only in channel catfish. Phylogenetic analyses suggested that four catfish TLR genes have duplicated copies in the genome, i.e., TLR4, TLR5, TLR8, and TLR20. Six fish-specific TLRs were identified, and the vast majority of these belong to the TLR11 subfamily. In healthy catfish tissues, most of the tested TLR genes were ubiquitously expressed although expression levels varied among the 11 tested tissues. We tested nine TLRs for their expression in response to Edwardsiella ictaluri infection. They were significantly up-regulated in the spleen and liver, but down-regulated in the head kidney, suggesting their involvement in the immune responses against the intracellular bacterial pathogen in a tissue-specific manner in catfish, perhaps through rapid migration of phagocytes to infection sites.

Catfish represent 12% of teleost or 6.3% of all vertebrate species, and are of enormous economic value. Here we report a high-quality reference genome sequence of channel catfish (Ictalurus punctatus), the major aquaculture species in the US. The reference genome sequence was validated by genetic mapping of 54,000 SNPs, and annotated with 26,661 predicted protein-coding genes. Through comparative analysis of genomes and transcriptomes of scaled and scaleless fish and scale regeneration experiments, we address the genomic basis for the most striking physical characteristic of catfish, the evolutionary loss of scales and provide evidence that lack of secretory calcium-binding phosphoproteins accounts for the evolutionary loss of scales in catfish. The channel catfish reference genome sequence, along with two additional genome sequences and transcriptomes of scaled catfishes, provide crucial resources for evolutionary and biological studies. This work also demonstrates the power of comparative subtraction of candidate genes for traits of structural significance.

Vertebrates including teleost fish have evolved an array of pathogen recognition receptors (PRRs) for detecting and responding to various pathogen-associated molecular patterns (PAMPs), including Toll-like receptors (TLRs), nucleotide-binding domain, leucine-rich repeat containing receptors (NLRs), and the retinoic acid inducible gene I (RIG-I) like receptors (RLRs). As a part of the series of studies targeted to characterize catfish PRRs, we described 22 NLR receptors in the sister contribution. Here in this study, we focused on cytosolic PRRs recognizing nucleotide pathogen-associated molecular patterns (PAMPs) of invading viruses, the retinoic acid-inducible gene I (RIG-I)-like receptors (RLR receptors). Three RLRs with DExD/H domain containing RNA helicases, retinoic acid inducible gene-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), were identified from channel catfish, Ictalurus punctatus. The catfish RIG-I encodes 937 amino acids that contains two CARDs, a DExDc, a HELICc and a RD domains. MDA5 encodes 1005 amino acids with all the domains identified for RIG-I. LGP2 encodes 677 amino acids that contain other domains but not the CARD domain at the N-terminus. Phylogenetic analyses of the three genes of catfish showed close clustering with their counterparts from other teleost fish. All the genes were found to be constitutively expressed in various tissues of catfish with minor variations. Channel catfish ovarian cells when infected with channel catfish virus showed significant increase in the transcript abundance of all the three genes. Further, RLR genes showed significant increases in expression in the liver tissue collected at different time-points after bacterial infection as well. The results indicate that the catfish RLRs may play important roles in antiviral and anti-bacterial immune responses.