Early divergent responses to virulent and attenuated vaccine isolates of Flavobacterium covae sp. nov. In channel catfish, Ictalurus punctatus.

Columnaris disease continues to inflict substantial losses among freshwater cultured species since its first description one hundred years ago. The experimental and anecdotal evidence suggests an expanded range and rising virulence of columnaris worldwide due to the warming global climate. The channel catfish (Ictalurus punctatus) are particularly vulnerable to columnaris. A recently developed live attenuated vaccine (17-23) for Flavobacterium columnare (now Flavobacterium covae sp. nov.) demonstrated superior protection for vaccinated catfish against genetically diverse columnaris isolates. In this study, we aimed to elucidate the molecular mechanisms and patterns of immune evasion and host manipulation linked to virulence by comparing gene expression changes in the host after the challenge with a virulent (BGSF-27) or live attenuated F. covae sp. nov. vaccine (17-23). Thirty-day-old fry were accordingly challenged with either virulent or vaccine isolates. Gill tissues were collected at 0 h (control), 1 h, and 2 h post-infection, which are two critical time points in early host-pathogen interactions. Transcriptome profiling of the gill tissues revealed a larger number (518) of differentially expressed genes (DEGs) in vaccine-exposed fish than those exposed to the virulent pathogen (321). Pathway analyses suggested potent suppression of early host immune responses by the virulent isolate through a higher expression of nuclear receptor corepressors (NCoR) responsible for antagonizing macrophage and T-cell signaling. Conversely, in vaccinated fry, we observed induction of Ca2+/calmodulin-dependent protein kinase II (CAMKII), responsible for clearing NCoR, and commensurate up-regulation of transcription factor AP-1 subunits, c-Fos, and c-Jun. As in mammalian systems, AP-1 expression was connected with a broad immune activation in vaccinated fry, including induction of CC chemokines, proteinases, iNOS, and IL-12b. Relatedly, divergent expression patterns of Src tyrosine kinase Lck, CD44, and CD28 indicated a delay or suppression of T-cell adhesion and activation in fry exposed to the virulent isolate. Broader implications of these findings will be discussed. The transcriptomic differences between virulent and attenuated bacteria may offer insights into how the host responds to the vaccination or infection and provide valuable knowledge to understand the early immune mechanisms of columnaris disease in aquaculture.

Immunological and biochemical changes in Pacific white shrimp, Litopenaeus vannamei, challenged with Vibrioparahaemolyticus.

Vibrio parahaemolyticus (Vpara) is the causative agent of Acute Hepatopancreatic Necrosis Disease (AHPND), or Early Mortality Syndrome (EMS) in shrimp. Shrimp, like other invertebrates, lack an adaptive immune system and depend solely on innate immunity against invading pathogens. To better understand the defense mechanisms of shrimp to this problematic pathogen, we evaluated the changes in hematology, immunology and biochemical values of the hemolymph from shrimp challenged with V. parahaemolyticus up to 8 days post-challenge. Thirty-six shrimp (12 g) were distributed in 9 tanks (75 L), divided into three groups (non-challenged, challenged with 5 × 102 cfu/shrimp and challenged with 1 × 103 cfu/shrimp) in triplicate. Pacific white shrimp, Litopenaeus vannamei, were administered an inoculum of V. parahaemolyticus under the shell between the 5th and 6th abdominal segment to assess cellular and humoral immune responses. Total hemocyte count (THC) significantly decreased in shrimp challenged with Vpara at 6 h, 12 h and 24 h-post infection. Hemocyte lysate phenoloxidase (PO) activity in Vpara-challenged shrimp at 48 h post challenge was significantly increased compared to that of control shrimp. No significant differences were observed in total plasma protein between plasma from control and Vpara-challenged shrimp. However, shrimp challenged with 5 × 102, and 1 × 103 cfu/shrimp had significantly lower hemocyanin at 6 h and 48 h sampling point, respectively. At 24 h post-challenge, the ≥140 kDa and 70 kDa bands from SDS-PAGE of hemocyanin-concentrated hemolymph lysate samples showed a higher and lower intensity, respectively, in Vpara-challenged group than those of the control group. Plasma from Vpara-challenged shrimp at 6 h and 12 h-post infection significantly suppressed V. parahaemolyticus growth. However, significantly less bacterial growth suppression was observed in plasma of shrimp challenged with higher dose compared to control shrimp at the 192 h post-challenge point. Plasma chemistry parameters did not significantly differ among treatments. The changes observed in hemolymph parameters may be useful indicators of the health status of shrimp.

Transcriptomic profiles of Florida pompano (Trachinotus carolinus) gill following infection by the ectoparasite Amyloodiniumocellatum.

The dinoflagellate Amyloodinium ocellatum is an important pathogenic parasite infecting cultured marine and brackish water fishes worldwide. This includes cultured Florida pompano (Trachinotus carolinus), which is one of the most desirable marine food fish with high economic value in the USA. A. ocellatum infects fish gills and causes tissue damage, increased respiratory rate, reduced appetite, and mortality, especially in closed aquaculture systems. This study mimicked the natural infection of A. ocellatum in cultured pompano and conducted a transcriptomic comparison of gene expression in the gills of control and A. ocellatum infected fish to explore the molecular mechanisms of infection. RNA-seq data revealed 604 differentially expressed genes in the infected fish gills. The immunoglobulin genes (including IgM/T) augmentation and IL1 inflammation suppression were detected after infection. Genes involved in reactive oxygen species mediating parasite killing were also highly induced. However, excessive oxidants have been linked to oxidative tissue damage and apoptosis. Correspondingly, widespread down-regulation of collagen genes and growth factor deprivation indicated impaired tissue repair, and meanwhile the key executor of apoptosis, caspase-3 was highly expressed (25.02-fold) in infected fish. The infection also influenced the respiratory gas sensing and transport genes and established hypoxic conditions in the gill tissue. Additionally, food intake and lipid metabolism were also affected. Our work provides the transcriptome sequencing of Florida pompano and provides key insights into the acute pathogenesis of A. ocellatum. This information can be utilized for designing optimal disease surveillance strategies, future selection for host resistance, and development of novel therapeutic measures.

The effect of piscidin antimicrobial peptides on the formation of Gram-negative bacterial biofilms.

Fish-derived antimicrobial peptides are an important part of the innate immune system due to their potent antimicrobial properties. Piscidins are a class of antimicrobial peptides first described in hybrid striped bass (Morone chrysops x Morone saxatilis) but have also been identified in many other fish species. Previous work demonstrated the broad antimicrobial activity of piscidins against Gram-negative and Gram-positive bacterial species. This study sought to determine the extent to which class I (striped bass piscidin 1, white bass piscidin 1 and striped bass/white bass piscidin 3) and class II (striped bass piscidin 4 and white bass piscidin 5) piscidins inhibit biofilm formation of different Gram-negative bacteria. In general, the class I and II piscidins demonstrate potent activity against Escherichia coli and Flavobacterium columnare biofilms. The class II piscidins showed more activity against E. coli and F. columnare isolates than did the class I piscidins. The piscidins in general were much less effective against inhibiting Aeromonas hydrophila and A. veronii biofilm growth. Only the class I piscidins showed significant growth inhibition among the Aeromonas spp. examined.

Compromised intestinal epithelial barrier induces adaptive immune compensation that protects from colitis.

Mice lacking junctional adhesion molecule A (JAM-A, encoded by F11r) exhibit enhanced intestinal epithelial permeability, bacterial translocation, and elevated colonic lymphocyte numbers, yet do not develop colitis. To investigate the contribution of adaptive immune compensation in response to increased intestinal epithelial permeability, we examined the susceptibility of F11r(-/-)Rag1(-/-) mice to acute colitis. Although negligible contributions of adaptive immunity in F11r(+/+)Rag1(-/-) mice were observed, F11r(-/-)Rag1(-/-) mice exhibited increased microflora-dependent colitis. Elimination of T cell subsets and cytokine analyses revealed a protective role for TGF-ß-producing CD4(+) T cells in F11r(-/-) mice. Additionally, loss of JAM-A resulted in elevated mucosal and serum IgA that was dependent upon CD4(+) T cells and TGF-ß. Absence of IgA in F11r(+/+)Igha(-/-) mice did not affect disease, whereas F11r(-/-)Igha(-/-) mice displayed markedly increased susceptibility to acute injury-induced colitis. These data establish a role for adaptive immune-mediated protection from acute colitis under conditions of intestinal epithelial barrier compromise.

Analysis of specific mRNA gene expression profiles as markers of egg and embryo quality for hybrid catfish aquaculture.

Despite best efforts to optimize reproduction, egg incubation, and larval performance in captivity, inconsistencies in hatchery fish production are still created by high variations in egg quality from individual females. In some hatchery species, egg quality and generation of viable embryos are correlated to abundances of specific mRNAs. Channel catfish females show considerable extremes in egg quality, causing inconsistencies in channel catfish, Ictalurus punctatus, female × blue catfish, Ictalurus furcatus, male hybrid fry production. The objectives of this study were to examine relative transcripts linked to egg and embryo quality and determine expression between low-hatch and high-hatch egg batches through early development (0, 24, 48, and 96 h post-fertilization; HPF). RNA was extracted from eggs/embryos of nine females (n = 4 high-quality, n = 5 low-quality) and Real-Time PCR was used to quantify relative gene expression. The transcripts assessed in this study perform critical cellular functions, including tubulin ß (tubb), cathepsin D (ctsd), cathepsin Z (ctsz), cathepsin B (ctsb), cyclin B (ccnb1), exportin-1 (xpo1), ring finger protein 213 (rnf213), glucocorticoid receptor-1 (GR-1), and heat shock protein 70 (hsp70). Relative gene expression of all transcripts except GR-1 and hsp70 were up-regulated in the high-hatch group and peaked at 48 HPF (neurulation stage), indicating the importance of these gene products at this threshold to normally progress until hatch. Due to lack of expression during earlier stages, maternally derived mRNAs for these genes do not seem to impact early embryonic development. Using mRNA markers as a selection mechanism for hatchery broodstock may lead to more high-hatch egg batches by reducing problems associated with poor egg quality.

Evidence that the stress hormone cortisol regulates biofilm formation differently among Flavobacterium columnare isolates.

The impact of cortisol on Flavobacterium columnare biofilm formation was explored. Firstly, the dynamics of biofilm formation by one highly (HV) and one low virulent (LV) F. columnare isolate with and without the stress hormone cortisol under microfluidic flow conditions was characterized. This to confirm that F. columnare cells could form biofilm under cortisol supplementation, and to compare the temporal and structural differences between different treatment groups. One trial revealed that in both isolates cell aggregates resembling biofilms occurred within 7-h post-inoculation. Consequently, cell clusters were sloughed away, followed by a rebuilding of bacterial cell aggregates, suggestive for a high spreading capacity. While the HV isolate revealed cell aggregates formed upstream at all time-points, for the LV isolate this was only seen upon cortisol supplementation. Secondly, the transcriptional effect of genes (gldK, gldL, gldM, gldN, sprA, sprE, sprT, and porV) belonging to the Type IX secretion system involved in gliding motility was investigated in planktonic and biofilm cells of a HV and LV isolate to which no, a low (LD) or high (HD) dose of cortisol was added. Significantly lower expression of gliding genes gldK, gldL, gldM and gldN, and of protein secretion regulator porV was seen in the LV isolate planktonic cells supplemented with a HD-cortisol. The LV isolate biofilm cells treated with the HD-cortisol showed a significant upregulation of sprT, encoding mobile surface adhesion important in bacterial colonization. This is the first evidence for the co-regulatory effect of cortisol on biofilm formation and F. columnare gliding gene expression.

Antimicrobial activity of the biopolymer chitosan against Streptococcus iniae.

The antimicrobial activity and mode of action of chitosan were evaluated against Streptococcus iniae, a pathogenic Gram-positive bacterium of fish worldwide. Cell proliferation kinetics were examined following exposure to varying concentrations of chitosan. The action of chitosan on S. iniae was also investigated by measuring agglutination activity, conductivity, and extracellular and intracellular bacterial adenosine triphosphate (ATP) levels. Chitosan exhibited antibacterial activity against S. iniae at concentrations of 0.1% and above and was lethal at a concentration of 0.4% and higher. The mechanism of antibacterial activity of chitosan at the inhibitory level of bacterial growth appears to hinge upon the interaction between chitosan and the oppositely charged bacterial surface. This interplay causes agglutination, which was readily observed grossly and microscopically. After interacting with the cell surface via adsorption, an efflux of intracellular ATP was documented, which suggests that chitosan disrupts the bacterial cell causing leakage of cytosolic contents and ultimately cell death. Results suggest chitosan may be worth evaluating as a natural alternative to antibiotic against S. iniae infection of fish.

l-rhamnose-binding lectins (RBLs) in Nile tilapia, Oreochromis niloticus: Characterization and expression profiling in mucosal tissues.

Rhamnose-binding lectins (RBLs) are crucial elements associated with innate immune responses to infections and have been characterized from a variety of teleost fishes. Given the importance of RBL in teleost fishes, we sought to study the diversity and expression profiles of RBLs in an important cultured fish, Nile tilapia (Oreochromis niloticus) following experimental infection with Streptococcus agalactiae, a major cause of streptococcosis in farmed tilapia. In this study, four predicted RBL genes were identified from Nile tilapia and were designated as OnRBL3a, OnRBL3b, OnRBL3c, and OnRBL3d. These OnRBLs were composed of two tandem-repeated type five carbohydrate recognition domains (CRDs), classified as type IIIc, and all clustered together phylogenetically. OnRBL-CRDs shared conserved topology of eight cysteine residues, characteristic peptide motifs of -YGR- and -DPC- (or -FGR- and -DTC-), and similar exon/intron organization. OnRBLs had the highest expression in immune-related tissues, gills, intestine or liver. However, the changes of OnRBL expression in the gills and intestine at 2 h, 4 h and 24 h post S. agalactiae challenge were modest, suggesting that tilapia may not mediate the entry or confront the infection of S. agalactiae through induction of RBL genes. The observed expression pattern may be related to the RBL type and CRD composition, S. agalactiae pathogenesis, the accessibility of ligands on the bacterial surface, and/or the species of fish. OnRBLs characterized in this study were the first RBL members identified in Nile tilapia and their characterization will expand our knowledge of RBLs in immunity.

Winter kill in intensively stocked channel catfish (Ictalurus punctatus): Coinfection with Aeromonas veronii, Streptococcus parauberis and Shewanella putrefaciens.

Unusual persistent natural mortality occurred in a floating in-pond raceway system intensively stocked with channel and hybrid catfish beginning in early November 2016 up until March 2017. The temperature during the period of outbreak ranged from 7.2 to 23.7°C. Gross examination of freshly dead and moribund fish revealed pale gills, slight abdominal distension and swollen inflamed vents. Comprehensive necropsy of 20 fish demonstrated vast amounts of bloody ascitic fluid in the coelomic cavity, visceral congestion, splenomegaly and pale friable livers but macroscopically normal kidneys, suggesting systemic bacterial infection. Bacterial cultures were initiated from skin, gills and major internal organs. Following incubation, a mixture of three bacterial colony phenotypes was observed on agar plates. Presumptive biochemical characterization of the isolates followed by 16S-rRNA sequence analysis resulted in the identification of Aeromonas veronii, Streptococcus parauberis and Shewanella putrefaciens. Channel catfish juveniles were experimentally infected with the recovered isolates to fulfil Koch's postulates. Moreover, an antibiogram was used to evaluate the susceptibility of the isolates to antimicrobial drugs approved for use in aquaculture. Aquaflor was used successfully for treatment. Here, we report bacterial coinfection lead by A. veronii and the first identification of S. parauberis and S. putrefaciens from cultured catfish in North America.

Influence of native catfish mucus on Flavobacterium columnare growth and proteolytic activity.

Flavobacterium columnare causes columnaris disease of farmed and wild freshwater fish. Skin mucus is an important factor in early stages of columnaris pathogenesis, albeit little studied. Our objectives were to (a) characterize the terminal glycosylation pattern (TGP) of catfish mucus, (b) determine the growth of F. columnare in formulated water (FW)-containing channel catfish (Ictalurus punctatus) or hybrid catfish (Ictalurus punctatus X Ictalurus furcatus) mucus and (c) examine extracellular protease activity of two F. columnare isolates differing in virulence. The TGP of catfish mucus by lectin binding was as follows: alpha-D-mannose/alpha-D-glucose >N-acetyl-beta-D-glucosamine >N-acetyl-beta-D-glucosamine/N-acetylneuraminic acid >N-acetyl-D-galactosamine >alpha-D-galactose/N-acetyl-alpha-D-galactosamine >beta-D-galactose = alpha-L-fucose. Virulence studies demonstrated isolate AL-02-36 was highly virulent in channel catfish fry (0.1 g) with cumulative mortality of 90%-100% versus 60% for isolate ALG-00-530 at equivalent doses (~3 × 106  CFU/ml); a similar result was observed in larger (0.7 g) catfish. In multiple experiments, F. columnare replicated (2-3 logs) and survived (28 days) in formulated water-containing catfish mucus. Highly virulent isolate AL-02-36 possessed at least 2.5- to fivefold higher protease activity following growth in mucus than the less virulent ALG-00-530. Flavobacterium columnare utilized catfish mucus as a nutrient source and mucus presence modulated extracellular protease production.

Testicular germ line cell identification, isolation, and transplantation in two North American catfish species.

Our aim was to transplant blue catfish germ line stem cells into blastulae of triploid channel catfish embryos to produce interspecific xenogenic catfish. The morphological structure of the gonads of blue catfish (Ictalurus furcatus) in ~ 90- to 100-day-old juveniles, two-year-old juveniles, and mature adults was studied histologically. Both oogonia (12-15 µm, diameter with distinct nucleus 7-8 µm diameter) and spermatogonia (12-15 µm, with distinct nucleus 6-7.5 µm diameter) were found in all ages of fish. The percentage of germ line stem cells was higher in younger blue catfish of both sexes. After the testicular tissue was trypsinized, a discontinuous density gradient centrifugation was performed using 70, 45, and 35% Percoll to enrich the percentage of spermatogonial stem cells (SSCs). Four distinct cell bands were generated after the centrifugation. It was estimated that 50% of the total cells in the top band were type A spermatogonia (diameter 12-15 µm) and type B spermatogonia (diameter 10-11 µm). Germ cells were confirmed with expression of vasa. Blastula-stage embryos of channel catfish (I. punctatus) were injected with freshly dissociated blue catfish testicular germ cells as donor cells for transplantation. Seventeen days after the transplantation, 33.3% of the triploid channel catfish fry were determined to be xenogenic catfish. This transplantation technique was efficient, and these xenogenic channel catfish need to be grown to maturity to verify their reproductive capacity and to verify that for the first time SSCs injected into blastulae were able to migrate to the genital ridge and colonize. These results open the possibility of artificially producing xenogenic channel catfish males that can produce blue catfish sperm and mate with normal channel catfish females naturally. The progeny would be all C × B hybrid catfish, and the efficiency of hybrid catfish production could be improved tremendously in the catfish industry.

Aquaculture genomics, genetics and breeding in the United States: current status, challenges, and priorities for future research.

Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, high-density and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries.Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop for Aquaculture Genomics, Genetics, and Breeding held in late March 2016 in Auburn, Alabama, with participants from all parts of the United States.

More than just antibodies: Protective mechanisms of a mucosal vaccine against fish pathogen Flavobacterium columnare.

A recently developed attenuated vaccine for Flavobacterium columnare has been demonstrated to provide superior protection for channel catfish, Ictalurus punctatus, against genetically diverse columnaris isolates. We were interested in examining the mechanisms of this protection by comparing transcriptional responses to F. columnare challenge in vaccinated and unvaccinated juvenile catfish. Accordingly, 58 day old fingerling catfish (28 days post-vaccination or unvaccinated control) were challenged with a highly virulent F. columnare isolate (BGSF-27) and gill tissues collected pre-challenge (0 h), and 1 h and 2 h post infection, time points previously demonstrated to be critical in early host-pathogen interactions. Following RNA-sequencing and transcriptome assembly, differential expression (DE) analysis within and between treatments revealed several patterns and pathways potentially underlying improved survival of vaccinated fish. Most striking was a pattern of dramatically higher basal expression of an array of neuropeptides (e.g. somatostatin), hormones, complement factors, and proteases at 0 h in vaccinated fish. Previous studies indicate these are likely the preformed mediators of neuroendocrine cells and/or eosinophilic granular (mast-like) cells within the fish gill. Following challenge, these elements fell to almost undetectable levels (>100-fold downregulated) by 1 h in vaccinated fish, suggesting their rapid release and/or cessation of synthesis following degranulation. Concomitantly, levels of pro-inflammatory cytokines (IL-1b, IL-8, IL-17) were induced in unvaccinated fish. In contrast, in vaccinated catfish, we observed widespread induction of genes needed for collagen deposition and tissue remodeling. Taken together, our results indicate an important component of vaccine protection in fish mucosal tissues may be the sensitization, proliferation and arming of resident secretory cells in the period between primary and secondary challenge.

Impact of oral and waterborne administration of rhamnolipids on the susceptibility of channel catfish (Ictalurus punctatus) to Flavobacterium columnare infection.

Flavobacterium columnare is the causative agent of columnaris disease and causes tremendous morbidity and mortality of farmed fish globally. Previously, we identified a potential lectin-mediator (a rhamnose-binding lectin; RBL1a) of F. columnare adhesion and showed higher RBL1a expression in susceptible channel catfish under basal conditions and following infection. Exposure of challenged fish to the carbohydrate ligand l-rhamnose just prior to a challenge substantially decreased columnaris mortality and pathogen adherence via the down-regulation of RBL1a. While highly effective in protecting fish from columnaris, l-rhamnose is prohibitively expensive, underscoring the need for alternative cost-effective sources of rhamnose for disease control. One such alternative may be microbially produced glycolipid compounds termed rhamnolipids (RLs), which feature abundant l-rhamnose moieties and are readily available from commercial sources. In the present study, we examined whether commercially available RLs (administered either by immersion or via feed) would function similarly to l-rhamnose in affording host protection against F. columnare. A four-week feeding trial with basal and RL top-coated diets (basal diet + RLs) was conducted in channel catfish fingerlings. Surprisingly, columnaris challenges revealed significantly lower survival following the 10 d challenge period in RL diet fed fish when compared with the basal treatment group (p < 0.001). In fish fed RLs, we observed a rapid and large-scale upregulation of RBL1a immediately after challenge combined with a suppression of mucin and lysozyme transcripts. Similarly, fish that were briefly pre-exposed to RLs by immersion and then challenged exhibited lower survival as compared to unexposed fish during a 4 d trial. In conclusion, RLs do not represent an alternative to rhamnose as an experimental treatment for protecting catfish from columnaris mortality. Further research is needed to find other affordable and efficacious alternative sources of l-rhamnose.

Ribosomal protein genes are highly enriched among genes with allele-specific expression in the interspecific F1 hybrid catfish.

Interspecific hybrids provide a rich source for the analysis of allele-specific expression (ASE). In this work, we analyzed ASE in F1 hybrid catfish using RNA-Seq datasets. While the vast majority of genes were expressed with both alleles, 7-8 % SNPs exhibited significant differences in allele ratios of expression. Of the 66,251 and 177,841 SNPs identified from the datasets of the liver and gill, 5420 (8.2 %) and 13,390 (7.5 %) SNPs were identified as significant ASE-SNPs, respectively. With these SNPs, a total of 1519 and 3075 ASE-genes were identified. Gene Ontology analysis revealed that genes encoding cytoplasmic ribosomal proteins (RP) were highly enriched among ASE genes. Parent-of-origin was determined for 27 and 30 ASE RP genes in the liver and gill, respectively. The results indicated that genes from both channel catfish and blue catfish were involved in ASE. However, each RP gene appeared to be almost exclusively expressed from only one parent, indicating that ribosomes in the hybrid catfish were in the "hybrid" form. Overall representation of RP transcripts among the transcriptome appeared lower in the F1 hybrid catfish than in channel catfish or blue catfish, suggesting that the "hybrid" ribosomes may work more efficiently for translation in the F1 hybrid catfish.

Expression profile analysis of two cathepsin S in channel catfish (Ictalurus punctatus) mucosal tissues following bacterial challenge.

Cathepsin S belongs to the papain family of cysteine protease, and is considered to play key roles in immune responses after bacterial challenge. However, despite the recognized importance of Cathepsin S in immunity, no studies have systematically characterized Cathepsin S in catfish. In this regard, here, we characterized the Cathepsin S gene family in channel catfish, and investigated their expression patterns following two different Gram-negative bacterial challenge. In the present study, two Cathepsin S genes (ctss and ctssa) were captured in channel catfish. In comparison to other species, the catfish Cathepsin S genes are highly conserved in their structural features. Phylogenetic analysis indicated the strongest phylogenetic relationship with zebrafish, which is consistent with their evolutional relationships. Tissue distribution analysis revealed that Cathepsin S genes were ubiquitously expressed in catfish tissues. Following bacterial infection, the Cathepsin S genes were significantly up-regulated at most time-points in mucosal surfaces, with an acute response post Edwardsiella ictaluri infection. Obviously, the expression profiles were quite distinct between two Cathepsin S genes, across the tissues and between pathogens, suggesting that Cathepsin S genes may exert disparate roles in mucosal immune responses. Our findings here, provide early insight into the immune functions of Cathepsin S in catfish; however, further studies are needed to determine the mechanisms of Cathepsin S for antigen presentation during inflammatory processes and innate host defense.

Galectins in channel catfish, Ictalurus punctatus: Characterization and expression profiling in mucosal tissues.

Galectins, a family of ß-galactoside-binding lectins with conserved CRDs, which can recognize the glycans on the surface of viruses, bacteria and protozoan parasites, are emerging as key players in many important pathological processes, including acute and chronic inflammatory diseases, autoimmunity and apoptosis. Although galectins have attracted great interest in mammals, they are still poorly-characterized in teleost. Previously, several studies have reported their high expression levels in mucosal tissues before and post infection. Given the important roles for galectins in mucosal immunity, therefore, we characterized the galectin gene family and profiled family member expression after challenge with two different Gram-negative bacterial pathogens. Here, twelve galectins genes were captured in channel catfish (Ictalurus punctatus), and phylogenetic analysis showed the strongest relationship to zebrafish and salmon, which is consistent with their phylogenetic relationships. Furthermore, the galectin genes were widely expressed in catfish tissues, while most of the galectin genes were strongly expressed in mucosal tissues (skin, gill and intestine). In addition, the expression profiles of galectins after bacterial infection varied depending on both pathogen and tissue type, suggesting that galectins may exert disparate functions or exhibit distinct tissue-selective roles in the host immune response to bacterial pathogens. Further studies are needed, however, to expand functional characterization and examine whether galectins may also play additional physiological roles in catfish immunity.

Effects of transgenic sterilization constructs and their repressor compounds on hatch, developmental rate and early survival of electroporated channel catfish embryos and fry.

Channel catfish (Ictalurus punctatus) embryos were electroporated with sterilization constructs targeting primordial germ cell proteins or with buffer. Some embryos then were treated with repressor compounds, cadmium chloride, copper sulfate, sodium chloride or doxycycline, to prevent expression of the transgene constructs. Promoters included channel catfish nanos and vasa, salmon transferrin (TF), modified yeast Saccharomyces cerevisiae copper transport protein (MCTR) and zebrafish racemase (RM). Knock-down systems were the Tet-off (nanos and vasa constructs), MCTR, RM and TF systems. Knock-down genes included shRNAi targeting 5' nanos (N1), 3' nanos (N2) or dead end (DND), or double-stranded nanos RNA (dsRNA) for overexpression of nanos mRNA. These constructs previously were demonstrated to knock down nanos, vasa and dead end, with the repressors having variable success. Exogenous DNA affected percentage hatch (% hatch), as all 14 constructs, except for the TF dsRNA, TF N1 (T), RM DND (C), vasa DND (C), vasa N1 (C) and vasa N2 (C), had lower % hatch than the control electroporated with buffer. The MCTR and RM DND (T) constructs resulted in delayed hatch, and the vasa and nanos constructs had minimal effects on time of hatch (P < 0.05). Cadmium chloride appeared to counteract the slow development caused by the TF constructs in two TF treatments (P < 0.05). The 4 ppt sodium chloride treatment for the RM system decreased % hatch (P < 0.05) and slowed development. In the case of nanos constructs, doxycycline greatly delayed hatch (P < 0.05). Adverse effects of the transgenes and repressors continued for several treatments for the first 6 days after hatch, but only in a few treatments during the next 10 days. Repressors and gene expression impacted the yield of putative transgenic channel catfish fry, and need to be considered and accounted for in the hatchery phase of producing transgenically sterilized catfish fry and their fertile counterparts. This fry output should be considered to ensure that sufficient numbers of transgenic fish are produced for future applications and for defining repressor systems that are the most successful.

Expression profiling analysis of immune-related genes in channel catfish (Ictalurus punctatus) skin mucus following Flavobacterium columnare challenge.

Fish are covered by a watery gel-mucus, mainly secreted by the goblet cells, serving as the physical and biochemical barrier between the external environment and the interior milieu, playing more important roles in fish that without scale. Despite the important roles of mucus in fish immunity, the knowledge of detailed molecular events happened during infection process is still limited. While most studies were focused on characterizing the protein and enzyme activities in the mucus following challenge, no studies have examined the gene expression profiles in fish mucus. In this regard, herein we carried out the first gene profiling analysis in catfish mucus using real-time PCR. Ten important immune-related genes were selected according to our previous studies. Their expression levels were examined in the early timepoints (namely, 1 h, 2 h, 4 h, 8 h, and 24 h) following Flavobacterium columnare challenge. Notably, expression levels of most of the selected genes were rapidly altered by the challenge. Seven genes were down-regulated, while only three genes were up-regulated. In addition, the gene expression patterns in mucus were very different from the mucosal surfaces (skin, gill and intestine) and the classical immune organs (liver, spleen and kidney). The unique expression patterns obtained here may be resulted from the great advantage of the large amount of attached bacteria in the mucus than the internal tissues, and resulted from the bacteria virulent actors to suppress the host immune response. Taken together, our results can expand our knowledge of fish mucosal immunity, and the un-lethal mucus sampling can provide early insight for developing the strategies for selection of disease resistant families and strains in catfish as well as other fish species.