Hypervirulent Aeromonas hydrophila (vAh) causes devastating damage in aquaculture worldwide. All vAh strains utilize myo-inositol (MI) as a sole carbon source prompted the previous study of the MI pathway in virulence. A range of gene-deletion mutants in the MI pathway were investigated through in vivo fish challenges to identify the insignificant role of MI pathway in virulence. However, the unexpected virulence of iolR mutants suggested the importance of IolR regulon as the transcriptional regulator for virulence. This study discerned the role of the IolR regulons on the pathogenesis of vAh. The A. hydrophila ML09-119 iolR gene was mutated to obtain different iolR expression levels. The results showed that increasing IolR expression caused decreased mortality in channel catfish 6 h post challenge, suggesting the role of IolR in repressing virulence gene transcription. A transcriptome analysis was conducted for ML09-119 and mutants. The results indicated that multiple putative genes involved in MI metabolism, fimbrial biogenesis, cell envelope integrity, and horizontal gene transfer might have IolRregulated transcription, with a palindromic sequence identified as the putative IolR binding site. Notably, when comparing phenotypes of iolR mutants of the channel catfish isolate ML09-119 and the carp isolate NJ-35, the regulation of biofilm formation and auto-aggregation were only observed for the carp isolate, which may reflect host-specific transcriptional regulation. Further study is necessary to understand the differences in the IolR regulon for carp and catfish isolates, as well as the contribution of each of the IolR-regulated genes to vAh pathogenesis.

Channel catfish, Ictalurus punctatus, is the most produced aquaculture species in the United States, although production levels have decreased since their peak in 2003. Myostatin (mstn) regulates skeletal muscle growth and has been identified as an important gene for increasing body weight in aquaculture species. In this study, the effects of CRISPR/Cas9 knockout of the mstn gene in channel catfish were investigated. Three sgRNAs targeting exon 1 of the channel catfish myostatin gene were microinjected with Cas9 protein in embryos. A total of 209 fish survived to 6 mph after microinjection of Cas9 and sgRNA targeting exon 1 of the mstn gene over the 3 yr experiment (2017-2019) with an average mutation rate of 58%. Successful generation of myostatin knockout (MSTN KO) F1 heterozygotes was achieved in 2019 by individually mating two pairs of control females with MSTN homozygous KO males. The offspring of both families inherited the mutation at a mean rate of 88%. Growth was generally higher in MSTN mutants than controls at all life stages and environments. P1 MSTN mutants were 88% larger than controls at the stocker stage (100 to 200 g) and 27% larger than controls at market size. Heterozygous F1 mutants were 218% larger than controls at the stocker stage. MSTN mutants had reduced overall expression levels of mstn compared to controls. When challenged with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish, MSTN mutants performed equally or better than controls. With a high mutation rate and inheritance as well as improved growth and disease resistance, using MSTN gene-edited channel catfish could greatly benefit commercial farms.

One of the major goals in aquaculture is to protect fish against infectious diseases as disease outbreaks could lead to economic losses if not controlled. Antimicrobial peptides (AMPs), a class of highly conserved peptides known to possess direct antimicrobial activities against invading pathogens, were evaluated for their ability to protect Channel Catfish Ictalurus punctatus and hybrid catfish (female Channel Catfish x male Blue Catfish I. furcatus) against infection caused by the fish pathogen Aeromonas hydrophila ML09-119. To identify effective peptides, the minimum inhibitory concentrations against bacterial pathogens Edwardsiella ictaluri S97-773, Edwardsiella piscicida E22-10, A. hydrophila ML09-119, Aeromonas veronii 03X03876, and Flavobacterium columnare GL-001 were determined in vitro. In general and overall, cathelicidins derived from alligator and sea snake exhibited more potent and rapid antimicrobial activities against the tested catfish pathogens as compared to cecropin and pleurocidin AMPs and ampicillin, the antibiotic control. When the peptides (2.5 mu g of peptide/g of fish) were injected into fish and simultaneously challenged with A. hydrophila through immersion, increased survival rates in Channel Catfish and hybrid catfish were observed in both cathelicidin (alligator and sea snake) treatments as compared to other peptides and the infected control (P < 0.001) with alligator cathelicidin being the overall best treatment. Bacterial numbers in the kidney and liver of Channel Catfish and hybrid catfish also decreased (P < 0.05) for cathelicidin-injected groups at 24 and 48 h after challenge infection. These results show the potential of cathelicidin to protect catfish against bacterial infections and suggest that an approach overexpressing the peptide in transgenic fish, which is the long-term goal of this research program, may provide a method of decreasing bacterial disease problems in catfish as delivering the peptides via individual injection or feeding would not be economically feasible.

Cathelicidins are a class of antimicrobial peptides (AMPs) known to possess rapid and direct antimicrobial activities against a variety of microorganisms. Recently identified cathelicidins derived from alligator and sea snake were found to be more effective in inhibiting microbial growth than other AMPs previously characterized. The ability of these two cathelicidins along with the peptides, cecropin and pleurocidin, to protect channel catfish (Ictalurus punctatus, Rafinesque) and hybrid catfish (I. punctatus ♀ × blue catfish, Ictalurus furcatus, Valenciennes ♂) against Edwardsiella ictaluri, one of the most prevalent pathogens affecting commercial catfish industry, was investigated. Cathelicidin-injected fish (50 µg ml-1  fish-1 ) that were simultaneously challenged with E. ictaluri through bath immersion at a concentration of ~1 × 106 CFU/ml had increased survival rates compared with other peptide treatments and the infected control. Bacterial numbers were also reduced in the liver and kidney of channel catfish and hybrid catfish in the cathelicidin treatments 24 hr post-infection. After 8 days of challenge, serum was collected to determine immune-related parameters such as bactericidal activity, lysozyme, serum protein, albumin and globulin. These immune-related parameters were significantly elevated in fish injected with the two cathelicidins as compared to other peptide treatments. These results indicate that cathelicidins derived from alligator and sea snake can stimulate immunity and enhance the resistance to E. ictaluri infection in channel catfish and hybrid catfish.

CRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model organisms. Here, we succeeded in integrating with high efficiency an exogenous alligator cathelicidin gene into a targeted non-coding region of channel catfish (Ictalurus punctatus) chromosome 1 using two different donor templates (synthesized linear dsDNA and cloned plasmid DNA constructs). We also tested two different promoters for driving the gene, zebrafish ubiquitin promoter and common carp β-actin promoter, harboring a 250-bp homologous region flanking both sides of the genomic target locus. Integration rates were found higher in dead fry than in live fingerlings, indicating either off-target effects or pleiotropic effects. Furthermore, low levels of mosaicism were detected in the tissues of P1 individuals harboring the transgene, and high transgene expression was observed in the blood of some P1 fish. This can be an indication of the localization of cathelicidin in neutrophils and macrophage granules as also observed in most antimicrobial peptides. This study marks the first use of CRISPR/Cas9 HDR for gene integration in channel catfish and may contribute to the generation of a more efficient system for precise gene integration in catfish and other aquaculture species, and the development of gene-edited, disease-resistant fish.

A hypervirulent A. hydrophila (vAh) pathotype has been identified as the etiologic agent involved in disease outbreaks in farmed carp and catfish species in China and the Southeastern United States, respectively. To assess the role of the LPS O-antigen in vAh virulence, the O-antigen ligase (waaL) and O-antigen polymerase (wzy) genes were deleted. While neither waaL nor wzy were found to be required for vAh virulence, a waaL mutant was found to have a polar effect on an adjacent group 4 capsule (gfc) genetic operon that was predicted to play a role in capsule assembly. Mutations in the gfc operon attenuated vAh virulence in channel catfish, particularly the mutant lacking gfcD that is predicted to encode an outer membrane protein required for capsular polysaccharide export. Furthermore, the vAh gfcD mutant was found to lack significant biofilm-forming capacity or buoyancy compared to wild-type vAh and induced an adaptive immune response that protected catfish from vAh challenge. This study indicates the importance of the capsular polysaccharide assembly process in the pathogenesis of this highly virulent A. hydrophila pathotype.

Background Drug-resistant fish pathogens can cause significant economic loss to fish farmers. Since 2012, florfenicol has become an approved drug for treating both septicemia and columnaris diseases in freshwater fish. Due to the limited drug options available for aquaculture, the impact of the therapeutical florfenicol treatment on the microbiota landscape as well as the resistome present in the aquaculture farm environment needs to be evaluated. Results Time-series metagenomic analyses were conducted to the aquatic microbiota present in the tank-based catfish production systems, in which catfish received standard therapeutic 10-day florfenicol treatment following the federal veterinary regulations. Results showed that the florfenicol treatment shifted the structure of the microbiota and reduced the biodiversity of it by acting as a strong stressor. Planctomycetes, Chloroflexi, and 13 other phyla were susceptible to the florfenicol treatment and their abundance was inhibited by the treatment. In contrast, the abundance of several bacteria belonging to the Proteobacteria, Bacteroidetes, Actinobacteria, and Verrucomicrobia phyla increased. These bacteria with increased abundance either harbor florfenicol-resistant genes (FRGs) or had beneficial mutations. The florfenicol treatment promoted the proliferation of florfenicol-resistant genes. The copy number of phenicol-specific resistance genes as well as multiple classes of antibiotic-resistant genes (ARGs) exhibited strong correlations across different genetic exchange communities ( p < 0.05), indicating the horizontal transfer of florfenicol-resistant genes among these bacterial species or genera. Florfenicol treatment also induced mutation-driven resistance. Significant changes in single-nucleotide polymorphism (SNP) allele frequencies were observed in membrane transporters, genes involved in recombination, and in genes with primary functions of a resistance phenotype. Conclusions The therapeutical level of florfenicol treatment significantly altered the microbiome and resistome present in catfish tanks. Both intra-population and inter-population horizontal ARG transfer was observed, with the intra-population transfer being more common. The oxazolidinone/phenicol-resistant gene optrA was the most prevalent transferred ARG. In addition to horizontal gene transfer, bacteria could also acquire florfenicol resistance by regulating the innate efflux systems via mutations. The observations made by this study are of great importance for guiding the strategic use of florfenicol, thus preventing the formation, persistence, and spreading of florfenicol-resistant bacteria and resistance genes in aquaculture.

Ictalurus punctatus (channel catfish) is an economically important farmed fish particularly in the southeastern United States. Aquaculture sustainability is threatened by disease pressure and the eutrophic conditions resulted from intensive fish farming. Previous research identified Bacillus velezensis strains that reduced mortality due to bacterial pathogens when used as a feed amendment. This study was conducted to determine the effects of B. velezensis-amended feed on catfish growth performance, pond water quality, and on the microbiomes in fish intestines and pond water. B. velezensis AP193 was evaluated in a ten-week pond trial, with four replicate ponds per probiotic treatment or control group. Feed amended with B. velezensis AP193 induced a 40.4% or 32.6% increase in growth relative to control feed in fingerling catfish that originated from aquaria or raceways, respectively. No significant differences were observed in the catfish intestinal microbiota or the pond microbiota due to probiotic-amended feed. The water quality was improved in ponds in which fish were fed with the probiotic-amended feed, as significant reductions were found in total phosphorus (19%), total nitrogen (43%) and nitrate (75%). These data suggest that B. velezensis AP193 can promote catfish growth and improve pond water quality when used as a feed amendment.

This study was conducted to evaluate the effects of probiotic-amended diets fed to juvenile Nile tilapia, Oreochromis niloticus, on growth and susceptibility to Streptococcus iniae infection. Fish (average weight 16.5 +/- 0.2 g) were fed five diets formulated with Bacillus subtilis strains SB3086, SB3295, SB3615, or AP193 either individually or in combination of strains SB3086 and SB3615 at a targeted concentration of approximately 4 x 10(7) colony-forming units (CFU)/g of feed or with a basal control diet with no additives for 21d. After the 21-d growth trial, no significant difference in growth performance was observed with any probiotic-amended diet. Results from serum bactericidal activity showed a significant difference between treatments and the control (P=0.0002), except for the SB3295-amended diet (P=0.9020). Lysozyme activity was also significantly different in fish fed probiotic diets from those fed control diet (P=0.0001). After 21d of feeding, fish were challenged with S. iniae by intraperitoneal injection at a dosage of 8 x 10(6) CFU per fish. Results from the challenge also showed a significant difference between treatments and control (P=0.0001). Overall, fish fed with strain SB3615 showed the lowest percent mortality (44.0 +/- 7.2%) and those fed the control diet showed the highest mortality (77.3 +/- 7.0%). The combined feeding with strains SB3086 and SB3615 did not result in any significant difference in reducing mortality because of S. iniae infection in juvenile Nile tilapia when compared with the individual probiotic treatments.

This study was conducted to evaluate the individual and combined effects of long-term feeding of diets containing two probiotic Bacillus subtilis group strains (Aqua NZ and AP193) and the prebiotic Previda ® , a commercial hemicellulose extract, on growth performance, immune parameters and Aeromonas hydrophila susceptibility of juvenile Nile tilapia, Oreochromis niloticus . Nile tilapia of average weight 7.47 ± 0.11 g were fed diets formulated with the probiotics and/or the prebiotic, or a control diet for 8 weeks and, subsequently, challenged with A. hydrophila by intragastric gavage at a dosage of 3.9 × 10 7 CFU per fish. Fish attained a mean weight of 59.5 ± 0.99 g at the end of the growth period. Under the conditions of the present trial, none of the diets significantly improved mean per cent weight gain ( P  =   0.70), thermal growth coefficient ( P  =   0.88) or feed conversion ratio ( P  =   0.87) of Nile tilapia. Except for the diet containing the prebiotic Previda ® only ( P  =   0.17), all other diets resulted in significantly higher fish survival compared to the control ( P  <   0.05). The combined effect of the prebiotic and probiotic strains emerged as the most important diet with respect to mortality reduction. The mean lysozyme and respiratory burst activities did not show any significant differences between treatments and control ( P  =   0.14 and 0.32, respectively). Thus, these probiotic strains used in this study have the potential to prevent disease due to A. hydrophila in O. niloticus .

This study was conducted to evaluate the individual and combined effects of long-term feeding of diets containing two probiotic Bacillus subtilis group strains (Aqua NZ and AP193) and the prebiotic Previda((R)), a commercial hemicellulose extract, on growth performance, immune parameters and Aeromonas hydrophila susceptibility of juvenile Nile tilapia, Oreochromis niloticus. Nile tilapia of average weight 7.47 +/- 0.11g were fed diets formulated with the probiotics and/or the prebiotic, or a control diet for 8weeks and, subsequently, challenged with A.hydrophila by intragastric gavage at a dosage of 3.9x10(7) CFU per fish. Fish attained a mean weight of 59.5 +/- 0.99g at the end of the growth period. Under the conditions of the present trial, none of the diets significantly improved mean per cent weight gain (P=0.70), thermal growth coefficient (P=0.88) or feed conversion ratio (P=0.87) of Nile tilapia. Except for the diet containing the prebiotic Previda((R)) only (P=0.17), all other diets resulted in significantly higher fish survival compared to the control (P<0.05). The combined effect of the prebiotic and probiotic strains emerged as the most important diet with respect to mortality reduction. The mean lysozyme and respiratory burst activities did not show any significant differences between treatments and control (P=0.14 and 0.32, respectively). Thus, these probiotic strains used in this study have the potential to prevent disease due to A.hydrophila in O.niloticus.

Lineages of hypervirulent Aeromonas hydrophila (vAh) are the cause of persistent outbreaks of motile Aeromonas septicemia in warm-water fishes worldwide. Over the last decade, this virulent lineage of A. hydrophila has resulted in annual losses of millions of tons of farmed carp and catfish in the People's Republic of China and the United States (US). Multiple lines of evidence indicate US catfish and Asian carp isolates of A. hydrophila affiliated with sequence type 251 (ST251) share a recent common ancestor. To address the genomic context for the putative intercontinental transfer and subsequent geographic spread of this pathogen, we conducted a core genome phylogenetic analysis on 61 Aeromonas spp. genomes, of which 40 were affiliated with A. hydrophila, with 26 identified as epidemic strains. Phylogenetic analyses indicate all ST251 strains form a coherent lineage affiliated with A. hydrophila. Within this lineage, conserved genetic loci unique to A. hydrophila were identified, with some genes present in consistently higher copy numbers than in non-epidemic A. hydrophila isolates. In addition, results from analyses of representative ST251 isolates support the conclusion that multiple lineages are present within US vAh isolated from Mississippi, whereas vAh isolated from Alabama appear clonal. This is the first report of genomic heterogeneity within US vAh isolates, with some Mississippi isolates showing closer affiliation with the Asian grass carp isolate ZC1 than other vAh isolated in the US. To evaluate the biological significance of the identified heterogeneity, comparative disease challenges were conducted with representatives of different vAh genotypes. These studies revealed that isolate ZC1 yielded significantly lower mortality in channel catfish, relative to Alabama and Mississippi vAh isolates. Like other Asian vAh isolates, the ZC1 lineage contains all core genes for a complete type VI secretion system (T6SS). In contrast, more virulent US isolates retain only remnants of the T6SS (clpB, hcp, vgrG, and vasH) which may have functional implications. Collectively, these results characterize a hypervirulent A. hydrophila pathotype that affects farmed fish on multiple continents.

Disease issues relevant to black bass populations arise from infectious as well as noninfectious etiologies. While disease outbreaks can occur via direct means, as is the case with primary pathogens, mortality events can also be linked to factors that disrupt the delicate balance between the environment, host, and pathogen. Indeed, black bass are found in a variety of geographies and habitats, and irrespective of their locale, fish are subject to environmental fluctuations, such as changes in dissolved oxygen, temperature, and water quality, as well as physical affronts, such as handling and confinement. These and other associated stressors can disrupt homeostasis and result in physiologic perturbations that are central to the pathophysiology of disease in black bass. Many pathogens that affect black bass are ubiquitous and opportunistic and commonly have limited impacts on populations as a whole unless a degradation of environmental conditions occurs that predispose fish to disease or exacerbate disease development. Examples include common aquatic bacterial pathogens (e.g., Aeromonas sp. and Flavobacterium columnare) and fungal and parasitic infestations, especially commensal protozoan parasites. In recent years, viral pathogens have been linked to large-scale fish mortalities in extensive, natural habitats as well as managed recreational impoundments. The underlying mechanisms behind these outbreaks remain largely undefined, yet significant concerns regarding biosecurity practices have surfaced due to the tremendous economic impacts that black bass fisheries support. Additionally, some grossly obvious phenotypic alterations of unknown etiologies (e.g., neoplasms and hyperpigmentation) may be indicative of environmental concerns that warrant further exploration.

The intensification of productivity in aquaculture has been accompanied by an increasing demand for environmentally friendly production. Ecological impacts including emergence of a large variety of pathogens and the development of resistant bacterial strains are growing. These impacts are in part due to the indiscriminate use of chemotherapeutic agents as a result of poor management practices in production cycles. There is a rapidly increasing literature pointing to the success of immunostimulants, plant products, and oral vaccines that can enhance growth; stimulate the innate, cellular, and/or humoral immune response; and control the number of pathogens that lead to fish diseases. It has been documented that supplemented diets may play an important role in improving immunity, health status, feed efficiency, nutrition, and growth performance of the host organism. Prebiotics are indigestible components in a diet that are metabolized by specific microorganisms and prove helpful for the growth and health of the host by selectively stimulating the growth of and/or activating the metabolism of one or a limited number of health-promoting bacteria in the intestinal tract. Probiotics are microorganisms or their products that exhibit health benefits to the host. Probiotics actively inhibit the colonization of potential pathogens in the digestive tract by antibiosis or by competition for nutrients and/or space, alteration of microbial metabolism, and/or the stimulation of host immunity. They also may stimulate appetite and improve nutrition by the production of vitamins, detoxification of compounds in the diet, and breakdown of indigestible components. There is accumulating evidence that probiotics are effective at inhibiting a wide range of fish pathogens, but the reasons for the inhibitions are not completely understood and evidence is often contradictory. However, the modes of action of these compounds are still considered to be hypotheses to be widely investigated. Dietary applications of prebiotics and probiotics may be combined to achieve a common objective in sustainable fish production. This commentary highlights the understanding of prebiotics, probiotics, and their combination, which provides pointers to the future of aquaculture.

ABSTRACT Since 2009, catfish farming in the southeastern United States has been severely impacted by a highly virulent and clonal population of Aeromonas hydrophila causing motile Aeromonas septicemia (MAS) in catfish. The possible origin of this newly emerged highly virulent A. hydrophila strain is unknown. In this study, we show using whole-genome sequencing and comparative genomics that A. hydrophila isolates from diseased grass carp in China and catfish in the United States have highly similar genomes. Our phylogenomic analyses suggest that U.S. catfish isolates emerged from A. hydrophila populations of Asian origin. Furthermore, we identified an A. hydrophila strain isolated in 2004 from a diseased catfish in Mississippi, prior to the onset of the major epidemic outbreaks in Alabama starting in 2009, with genomic characteristics that are intermediate between those of the Asian and Alabama fish isolates. Investigation of A. hydrophila strain virulence demonstrated that the isolate from the U.S. catfish epidemic is significantly more virulent to both channel catfish and grass carp than is the Chinese carp isolate. This study implicates the importation of fish or fishery products into the United States as the source of highly virulent A. hydrophila that has caused severe epidemic outbreaks in United States-farmed catfish and further demonstrates the potential for invasive animal species to disseminate bacterial pathogens worldwide. IMPORTANCE Catfish aquaculture farming in the southeastern United States has been severely affected by the emergence of virulent Aeromonas hydrophila responsible for epidemic disease outbreaks, resulting in the death of over 10 million pounds of catfish. Because the origin of this newly emerged A. hydrophila strain is unknown, this study used a comparative genomics approach to conduct a phylogenomic analysis of A. hydrophila isolates obtained from the United States and Asia. Our results suggest that the virulent isolates from United States-farmed catfish have a recent common ancestor with A. hydrophila isolates from diseased Asian carp. We have also observed that an Asian carp isolate, like recent U.S. catfish isolates, is virulent in catfish. The results from this study suggest that the highly virulent U.S. epidemic isolates emerged from an Asian source and provide another example of the threat that invasive species pose in the dissemination of bacterial pathogens.

A feeding trial was conducted to investigate the effects of dietary administration of probiotic with Bacillus subtilis, Aspergillus oryzae and Saccharomyces cerevisiae on growth, innate immune response, Hemato-immunological parameters and disease resistance of Nile tilapia, Oreochromis niloticus. Animals were distributed in three equal groups, each of five replicates and received one of the following experimental diets for four weeks: Control, non-supplemented diet; 5 g kg(-1) probiotic mixture (B. subtilis 1.5 x 10(9) CFU g(-1), S. cerevisiae 10(9) CFU g(-1) and A. oryzae 2 x 10(9) CPU g(-1)); and 10 g kg(-1) probiotic mixture (B. subtilis 3.0 x 10(9) CFU g(-1), S. cerevisiae 2.0 x 10(9) CPU g(-1) and A. oryzae 4.0 x 10(9) CFU g(-1)). The respiratory burst activity, white blood cells and hematological parameters were evaluated after four, five and six weeks of feeding. At the end of the growth trial, fish were sampled for intestinal microbiology and challenged by intraperitoneal injection of LD50 concentration of Aeromonas hydrophila and Streptococcus iniae. Mortality was recorded for the following 3 weeks. Results showed that administration of the probiotic had no significant effect on the growth rates of Nile tilapias, although the fish fed probiotics had better feed conversion. Respiratory burst activity, erythrocyte fragility and levels of white blood cells were significantly improved in tilapias fed diet supplemented with probiotic levels (P < 0.05), which may exhibit up-regulating effects on tilapia immune parameters. The cumulative mortality after A. hydrophila and S. iniae challenge decreased in tilapias fed with probiotic (P < 0.05). The present study demonstrated the potential of B. subtilis, S. cerevisiae and A. oryzae combined as beneficial dietary probiotic in juvenile O. niloticus. (C) 2014 Elsevier Ltd. All rights reserved.

The availability of African lungfish ( Protopterus aethiopicus ) in many communities in Uganda is declining. Indigenous efforts to culture this fish usually produce poor yields and depend on feeding fish fry, minced meat, and leftover food. This study evaluates three formulated diets (diet- 1, diet-2, diet-3) fed to wild caught lungfish fingerlings reared in indoor tanks for 77 days. Experimental fish gradually accepted sinking pellets, and marginal increases in average body weight were observed. Mean (± SE) final weight (15.86 ± 0.80 g) for fish fed on diet-3 was significantly higher (p 0.05), ranging from 1.61 ± 0.26 to 2.07 ± 0.11. Survivals after an 11-week culture were relatively low (< 60%), but generally increased (R2 = 0.667, P = 0.007) with increasing dietary proteins. Diet-3 had a significant higher survival rate (p< 0.05) than diet- 1 and diet-2. Significant growth performance was attained with diet-3. This study demonstrated that sinking fish feed pellets can be used to culture wild-caught African fingerlings in captivity. Key words : African lungfish, aquaculture, exogenous feeds

BACKGROUND:Calpains, a superfamily of intracellular calcium-dependent cysteine proteases, are involved in the cytoskeletal remodeling and wasting of skeletal muscle. Calpains are generated as inactive proenzymes which are activated by N-terminal autolysis induced by calcium-ions.METHODOLOGY/PRINCIPAL FINDINGS:In this study, we characterized the full-length cDNA sequences of three calpain genes, clpn1, clpn2, and clpn3 in channel catfish, and assessed the effect of nutrient restriction and subsequent re-feeding on the expression of these genes in skeletal muscle. The clpn1 cDNA sequence encodes a protein of 704 amino acids, Clpn2 of 696 amino acids, and Clpn3 of 741 amino acids. Phylogenetic analysis of deduced amino acid sequences indicate that catfish Clpn1 and Clpn2 share a sequence similarity of 61%; catfish Clpn1 and Clpn3 of 48%, and Clpn2 and Clpn3 of only 45%. The domain structure architectures of all three calpain genes in channel catfish are similar to those of other vertebrates, further supported by strong bootstrap values during phylogenetic analyses. Starvation of channel catfish (average weight, 15-20 g) for 35 days influenced the expression of clpn1 (2.3-fold decrease, P<0.05), clpn2 (1.3-fold increase, P<0.05), and clpn3 (13.0-fold decrease, P<0.05), whereas the subsequent refeeding did not change the expression of these genes as measured by quantitative real-time PCR analysis. Calpain catalytic activity in channel catfish skeletal muscle showed significant differences only during the starvation period, with a 1.2- and 1.4- fold increase (P<0.01) after 17 and 35 days of starvation, respectively.CONCLUSION/SIGNIFICANCE:We have assessed that fasting and refeeding may provide a suitable experimental model to provide us insight into the role of calpains during fish muscle atrophy and how they respond to changes in nutrient supply.

To investigate the molecular basis of the emergence of Aeromonas hydrophila responsible for an epidemic outbreak of motile aeromonad septicemia of catfish in the Southeastern United States, we sequenced 11 A. hydrophila isolates that includes five reference and six recent epidemic isolates. Comparative genomics revealed that recent epidemic A. hydrophila isolates are highly clonal, whereas reference isolates are greatly diverse. We identified 55 epidemic-associated genetic regions with 313 predicted genes that are present in epidemic isolates but absent from reference isolates and 35% of these regions are located within genomic islands, suggesting their acquisition through lateral gene transfer. The epidemic-associated regions encode predicted prophage elements, pathogenicity islands, metabolic islands, fitness islands and genes of unknown functions, and 34 of the genes encoded in these regions were predicted as virulence factors. We found two pilus biogenesis gene clusters encoded within predicted pathogenicity islands. A functional metabolic island that encodes a complete pathway for myo-inositol catabolism was evident by the ability of epidemic A. hydrophila isolates to use myo-inositol as a sole carbon source. Testing of A. hydrophila field isolates found a consistent correlation between myo-inositol utilization as a sole carbon source and the presence of an epidemic-specific genetic marker. All epidemic isolates and one reference isolate shared a novel O-antigen cluster. Altogether we identified four different O-antigen biosynthesis gene clusters within the 11 sequenced A. hydrophila genomes. Our study reveals new insights into the evolutionary changes that have resulted in the emergence of recent epidemic A. hydrophila strains.