Novel lytic bacteriophage AhFM11 as an effective therapy against hypervirulent Aeromonas hydrophila.

Several farmed fish species, including carps, tilapia, salmon, and catfish, have experienced significant economic losses in aquaculture due to motile Aeromonas septicemia caused by Aeromonas hydrophila. In the present study, a novel lytic bacteriophage infecting hypervirulent Aeromonas hydrophila (vAh) was isolated and characterized. This is the first report of a phage against vAh. Phage AhFM11 demonstrated lytic activity against both vAh strains and the A. hydrophila reference strain ATCC 35654. The AhFM11 genome was sequenced and assembled, comprising 168,243 bp with an average G + C content of 41.5%. The genome did not harbor any antibiotic resistance genes. Genomic information along with transmission electron microscopy revealed that phage AhFM11 belongs to the Straboviridae family. Therapeutic application of monophage AhFM11 in fish showed 100% survival in injection, 95% in immersion and 93% in oral feeding of phage top-coated feed. Fish and chicken meat spiked with A. hydrophila and phage showed significant reduction of A. hydrophila. These findings support that phage AhFM11 can be used as a biocontrol agent against vAh as an alternative to antibiotics.

Whole-Genome Sequencing and Annotation of Aeromonas veronii Isolates from Channel Catfish.

The genomes of seven Aeromonas veronii strains isolated from tissues of healthy or diseased channel catfish obtained from Alabama, USA, fish farms were sequenced and annotated. These genome sequences will enable comparative analyses to determine the roles these bacteria play in catfish aquaculture and the development of new preventative or management strategies.

Transcriptome analysis and immune gene expression of channel catfish (Ictalurus punctatus) fed diets with inclusion of frass from black soldier fly larvae.

The larval waste, exoskeleton shedding, and leftover feed components of the black soldier fly and its larvae make up the by-product known as frass. In this study, we subjected channel catfish (Ictalurus punctatus) to a 10-week feeding trial to assess how different dietary amounts of frass inclusion would affect both systemic and mucosal tissue gene expression, especially in regard to growth and immune-related genes. Fish were divided in quadruplicate aquaria, and five experimental diets comprising 0, 50, 100, 200, and 300 g of frass per kilogram of feed were fed twice daily. At the end of the trial, liver, head kidney, gill, and intestine samples were collected for gene expression analyses. First, liver and intestine samples from fish fed with a no frass inclusion diet (control), low-frass (50 g/kg) inclusion diet, or a high-frass (300 g/kg) inclusion diet were subjected to Illumina RNA sequencing to determine global differential gene expression among diet groups. Differentially expressed genes (DEGs) included the upregulation of growth-related genes such as glucose-6-phosphatase and myostatin, as well as innate immune receptors and effector molecules such as toll-like receptor 5, apolipoprotein A1, C-type lectin, and lysozyme. Based on the initial screenings of low/high frass using RNA sequencing, a more thorough evaluation of immune gene expression of all tissues sampled, and all levels of frass inclusion, was further conducted. Using targeted quantitative PCR panels for both innate and adaptive immune genes from channel catfish, differential expression of genes was identified, which included innate receptors (TLR1, TLR5, TLR9, and TLR20A), proinflammatory cytokines (IL-1ß type a, IL-1ß type b, IL-17, IFN-γ, and TNFα), chemokines (CFC3 and CFD), and hepcidin in both systemic (liver and head kidney) and mucosal (gill and intestine) tissues. Overall, frass from black soldier fly larvae inclusion in formulated diets was found to alter global gene expression and activate innate and adaptive immunity in channel catfish, which has the potential to support disease resistance in this species in addition to demonstrated growth benefits.

Hepatic transcriptome analyses of juvenile white bass (Morone chrysops) when fed diets where fish meal is partially or totally replaced by alternative protein sources.

White bass (Morone chrysops) are a popular sportfish throughout the southern United States, and one parent of the commercially-successful hybrid striped bass (M. chrysops ♂ x M. saxatilis ♀). Currently, white bass are cultured using diets formulated for other carnivorous fish, such as largemouth bass (Micropterus salmoides) or hybrid striped bass and contain a significant percentage of marine fish meal. Since there are no studies regarding the utilization of alternative proteins in this species, we evaluated the global gene expression of white bass fed diets in which fish meal was partially or totally replaced by various combinations of soybean meal, poultry by-product meal, canola meal, soy protein concentrate, wheat gluten, or a commercial protein blend (Pro-Cision™). Six isonitrogenous (40% protein), isolipidic (11%), and isocaloric (17.1 kJ/g) diets were formulated to meet the known nutrient and energy requirements of largemouth bass and hybrid striped bass using nutrient availability data for most of the dietary ingredients. One of the test diets consisted exclusively of plant protein sources. Juvenile white bass (40.2 g initial weight) were stocked into a flow-through aquaculture system (three tanks/diet; 10 fish/tank) and fed the test diets twice daily to satiation for 60 days. RNA sequencing and bioinformatic analyses revealed significant differentially expressed genes between all test diets when compared to fish meal control. A total of 1,260 differentially expressed genes were identified, with major ontology relating to cell cycle and metabolic processes as well as immune gene functions. This data will be useful as a resource for future refinements to moronid diet formulation, as marine fish meal becomes limiting and plant ingredients are increasingly added as a reliable protein source.

Differential susceptibility of white bass (Morone chrysops), striped bass (Morone saxatilis) and hybrid striped bass (M. chrysops × M. saxatilis) to Flavobacterium columnare and effects of mucus on bacterial growth and biofilm development.

Columnaris disease generates substantial losses of many freshwater fish species; one is the hybrid striped bass. The ubiquitous aquatic bacterium Flavobacterium columnare can be highly effective in biofilm formation on fish skin and gills. Previous research showed a difference between columnaris disease susceptibility of hybrid striped bass (Morone saxatilis × M. chrysops) and white bass (M. chrysops). To understand these differential susceptibilities and possible mucosal relationship, we assessed total bacterial growth and biofilm formation with mucus derived from each moronid parental species: white bass and striped bass (M. saxatilis). Differential susceptibility was confirmed of the other parent species, the striped bass (M. saxatilis). In addition to intraspecies investigations, individual hybrid striped bass mucosal affects were also studied for deferential responses to bacterial growth and biofilm formation. Species- and concentration-dependent differences were detected in the total growth of the bacteria to host mucus. Our data suggest that bass mucus can significantly affect biofilm formation with the F. columnare isolate tested. There appears to be a correlation between the bacteria's response of growth and biofilms and bass species susceptibility. This study provides insight into our understanding of the host-pathogen interaction between F. columnare and moronids.

Second-generation genetic linkage map of catfish and its integration with the BAC-based physical map.

Construction of high-density genetic linkage maps is crucially important for quantitative trait loci (QTL) studies, and they are more useful when integrated with physical maps. Such integrated maps are valuable genome resources for fine mapping of QTL, comparative genomics, and accurate and efficient whole-genome assembly. Previously, we established both linkage maps and a physical map for channel catfish, Ictalurus punctatus, the dominant aquaculture species in the United States. Here we added 2030 BAC end sequence (BES)-derived microsatellites from 1481 physical map contigs, as well as markers from singleton BES, ESTs, anonymous microsatellites, and SNPs, to construct a second-generation linkage map. Average marker density across the 29 linkage groups reached 1.4 cM/marker. The increased marker density highlighted variations in recombination rates within and among catfish chromosomes. This work effectively anchored 44.8% of the catfish BAC physical map contigs, covering ~52.8% of the genome. The genome size was estimated to be 2546 cM on the linkage map, and the calculated physical distance per centimorgan was 393 Kb. This integrated map should enable comparative studies with teleost model species as well as provide a framework for ordering and assembling whole-genome scaffolds.

Identification and characterization of matrix metalloproteinase-13 sequence structure and expression during embryogenesis and infection in channel catfish (Ictalurus punctatus).

Matrix metalloproteinase-13 (MMP-13), referred to as collagenase-3, is a proteolytic enzyme that plays a key role in degradation and remodelling of host extracellular matrix proteins. The objective of this study was to characterize the MMP-13 gene in channel catfish, and to determine its pattern of expression in various healthy tissues and during embryogenesis. Since MMP-13 has been shown to have importance in tissue remodelling and some pathological processes, we further studied its involvement in the defense responses of catfish after bacterial infection. The channel catfish MMP-13 cDNA contains an open reading frame of 1416bp encoding 471 amino acids. Using RT-PCR analysis, MMP-13 was widely expressed in various health tissues. Using quantitative real-time PCR analysis, expression of MMP-13 gene was up-regulated by bacterial infection. During normal embryological development, MMP-13 expression was slightly increased in the first day post-fertilization and sharply up-regulated from 1-day post-fertilization through hatching.

Molecular characterization of complement factor I reveals constitutive expression in channel catfish.

The complement system in vertebrates plays a crucial role in immune defense via recognition and removal of pathogens. Complement is tightly regulated by a group of both soluble and cell-associated proteins. Complement factor I is a soluble serine protease that regulates multiple pathways in complement activation. In this work, a complement factor I transcript was isolated and sequenced from channel catfish (Ictalurus punctatus) liver after screening expressed sequence tags. The full-length cDNA is comprised of 2284bp in length, encoding a polypeptide of 668 amino acids. The complement factor I protein was found to be well conserved, with similar domain structures and architecture from fish to mammals. The catfish complement factor I exists as a single-copied gene in the catfish genome. Expression analysis revealed that the catfish complement factor I is constitutively expressed in all tissues and leukocyte cell lines tested, indicating its importance as a regulatory enzyme throughout channel catfish. While expression of complement factor I is often found to be in the liver in mammals, it is constitutively expressed in channel catfish and carp throughout in various tissues and organs.

NOD-like subfamily of the nucleotide-binding domain and leucine-rich repeat containing family receptors and their expression in channel catfish.

The NLRs (nucleotide-binding domain and leucine-rich repeat containing family receptors) are a recently identified family of pattern recognition receptors in vertebrates. Several subfamilies of NLRs have been characterized in human, mouse, and zebrafish, but studies of NLRs in other species, especially teleost species, have been lacking. Here we report characterization of five NLRs from channel catfish: NOD1, NOD2, NLRC3, NLRC5, and NLRX1. Structural analysis indicated that the genes were organized in a similar fashion as in the mammals and in zebrafish. Phylogenetic analysis suggested that they were orthologous to the NOD-like subfamily of NLRs. All five NOD-like genes exist as a single copy gene in the catfish genome. Hybridization of gene-specific probes allowed mapping of three NLR genes to the catfish physical map, laying a foundation for genome characterization and for establishing orthologies with NLR genes from other species. These genes are widely expressed in various tissues and leukocyte cell lines. While the majority of the NLR genes appeared to be constitutively expressed, NOD1 was induced after infection with a bacterial pathogen, Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC), suggesting its involvement in immunity against the intracellular pathogen.

Transcriptomic profiling of Ichthyophthirius multifiliis reveals polyadenylation of the large subunit ribosomal RNA.

Polyadenylation of eukaryotic transcripts is usually restricted to mRNA, providing transcripts with stability from degradation by nucleases. Conversely, an RNA degradation pathway can be signaled through poly (A) tailing in prokaryotic, archeal, and organellar biology. Recently polyadenylated transcripts have also been discovered in rRNA in some eukaryotes including humans and yeast. Here we report the discovery of polyadenylated rRNAs in the ciliate teleost parasite Ichthyophthirius multifiliis, an important fish pathogen. Through large-scale analysis of ESTs, a large contig composed of the 28S rRNA with poly (A) tails was identified. Analysis using multiple sequence alignments revealed four potential polyadenylation sites including three internal regions and the 3? end of the rRNA. Further analysis using a polyadenylation test, re-sequencing, and gene-specific PCR using primers flanking the presumed poly (A) sites confirmed the presence of polyadenylated rRNA in this parasite. The functions of polyadenylation of rRNA in this organism are largely unknown at present, but the presence of internal polyadenylation sites, along with the presence of truncated segments of the rRNA, may suggest a role of the polyadenylation in the degradation pathway, a function typical of prokaryotes, archaea, and organelles. These results are in congruence with reports of a similar phenomenon in humans and yeast.

Generation and analysis of expressed sequence tags from the ciliate protozoan parasite Ichthyophthirius multifiliis.

BACKGROUND: The ciliate protozoan Ichthyophthirius multifiliis (Ich) is an important parasite of freshwater fish that causes 'white spot disease' leading to significant losses. A genomic resource for large-scale studies of this parasite has been lacking. To study gene expression involved in Ich pathogenesis and virulence, our goal was to generate expressed sequence tags (ESTs) for the development of a powerful microarray platform for the analysis of global gene expression in this species. Here, we initiated a project to sequence and analyze over 10,000 ESTs. RESULTS: We sequenced 10,368 EST clones using a normalized cDNA library made from pooled samples of the trophont, tomont, and theront life-cycle stages, and generated 9,769 sequences (94.2% success rate). Post-sequencing processing led to 8,432 high quality sequences. Clustering analysis of these ESTs allowed identification of 4,706 unique sequences containing 976 contigs and 3,730 singletons. These unique sequences represent over two million base pairs (~10% of Plasmodium falciparum genome, a phylogenetically related protozoan). BLASTX searches produced 2,518 significant (E-value < 10-5) hits and further Gene Ontology (GO) analysis annotated 1,008 of these genes. The ESTs were analyzed comparatively against the genomes of the related protozoa Tetrahymena thermophila and P. falciparum, allowing putative identification of additional genes. All the EST sequences were deposited by dbEST in GenBank (GenBank: EG957858-EG966289). Gene discovery and annotations are presented and discussed. CONCLUSION: This set of ESTs represents a significant proportion of the Ich transcriptome, and provides a material basis for the development of microarrays useful for gene expression studies concerning Ich development, pathogenesis, and virulence.