One-step knock-in of two antimicrobial peptide transgenes at multiple loci of catfish by CRISPR/Cas9-mediated multiplex genome engineering.

CRISPR/Cas9-mediated multiplex genome editing (MGE) conventionally uses multiple single-guide RNAs (sgRNAs) for gene-targeted mutagenesis via the non-homologous end joining (NHEJ) pathway. MGE has been proven to be highly efficient for functional gene disruption/knockout (KO) at multiple loci in mammalian cells or organisms. However, in the absence of a DNA donor, this approach is limited to small indels without transgene integration. Here, we establish the linear double-stranded DNA (dsDNA) and double-cut plasmid (dcPlasmid) combination-assisted MGE in channel catfish (Ictalurus punctatus), allowing combinational deletion mutagenesis and transgene knock-in (KI) at multiple sites through NHEJ/homology-directed repair (HDR) pathway in parallel. In this study, we used single-sgRNA-based genome editing (ssGE) and multi-sgRNA-based MGE (msMGE) to replace the luteinizing hormone (lh) and melanocortin-4 receptor (mc4r) genes with the cathelicidin (As-Cath) transgene and the myostatin (two target sites: mstn1, mstn2) gene with the cecropin (Cec) transgene, respectively. A total of 9000 embryos were microinjected from three families, and 1004 live fingerlings were generated and analyzed. There was no significant difference in hatchability (all P > 0.05) and fry survival (all P > 0.05) between ssGE and msMGE. Compared to ssGE, CRISPR/Cas9-mediated msMGE assisted by the mixture of dsDNA and dcPlasmid donors yielded a higher knock-in (KI) efficiency of As-Cath (19.93 %, [59/296] vs. 12.96 %, [45/347]; P = 0.018) and Cec (22.97 %, [68/296] vs. 10.80 %, [39/361]; P = 0.003) transgenes, respectively. The msMGE strategy can be used to generate transgenic fish carrying two transgenes at multiple loci. In addition, double and quadruple mutant individuals can be produced with high efficiency (36.3 % âˆ¼ 71.1 %) in one-step microinjection. In conclusion, we demonstrated that the CRISPR/Cas9-mediated msMGE allows the one-step generation of simultaneous insertion of the As-Cath and Cec transgenes at four sites, and the simultaneous disruption of the lh, mc4r, mstn1 and mstn2 alleles. This msMGE system, aided by the mixture donors, promises to pioneer a new dimension in the drive and selection of multiple designated traits in other non-model organisms.

CRISPR/Cas9-mediated knock-in of masu salmon (Oncorhyncus masou) elongase gene in the melanocortin-4 (mc4r) coding region of channel catfish (Ictalurus punctatus) genome.

Channel catfish, Ictalurus punctatus, have limited ability to synthesize Ω-3 fatty acids. The ccßA-msElovl2 transgene containing masu salmon, Oncorhynchus masou, elongase gene driven by the common carp, Cyprinus carpio, ß-actin promoter was inserted into the channel catfish melanocortin-4 receptor (mc4r) gene site using the two-hit two-oligo with plasmid (2H2OP) method. The best performing sgRNA resulted in a knockout mutation rate of 92%, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%. Fish containing both the ccßA-msElovl2 transgene knock-in and mc4r knockout (Elovl2) were 41.8% larger than controls at 6 months post-hatch (p = 0.005). Mean eicosapentaenoic acid (EPA, C20:5n-3) levels in Elov2 mutants and mc4r knockout mutants (MC4R) were 121.6% and 94.1% higher than in controls, respectively (p = 0.045; p = 0.025). Observed mean docosahexaenoic acid (DHA, C22:6n-3) and total EPA + DHA content was 32.8% and 45.1% higher, respectively, in Elovl2 transgenic channel catfish than controls (p = 0.368; p = 0.025). To our knowledge this is the first example of genome engineering to simultaneously target transgenesis and knock-out a gene in a commercially important aquaculture species for multiple improved performance traits. With a high transgene integration rate, improved growth, and higher omega-3 fatty acid content, the use of Elovl2 transgenic channel catfish appears beneficial for application on commercial farms.

Gene Editing of the Follicle-Stimulating Hormone Gene to Sterilize Channel Catfish, Ictalurus punctatus, Using a Modified Transcription Activator-like Effector Nuclease Technology with Electroporation.

Follicle-stimulating hormone (fsh) plays an important role in sexual maturation in catfish. Knocking out the fsh gene in the fish zygote should suppress the reproduction of channel catfish (Ictalurus punctatus). In this study, transcription activator-like effector nuclease (TALEN) plasmids targeting the fsh gene were electroporated into fertilized eggs with the standard double electroporation technique. Targeted fsh cleavage efficiency was 63.2% in P1fsh-knockout catfish. Ten of fifteen (66.7%) control pairs spawned, and their eggs had 32.3-74.3% average hatch rates in 2016 and 2017. Without hormone therapy, the spawning rates of P1 mutants ranged from 33.3 to 40.0%, with an average egg hatching rate of 0.75%. After confirmation of the low fertility of P1 mutants in 2016, human chorionic gonadotropin (HCG) hormone therapy improved the spawning rates by 80% for female mutants and 88.9% for male mutants, and the mean hatch rate was 35.0% for F1 embryos, similar to that of the controls (p > 0.05). Polymerase chain reaction (PCR) identification showed no potential TALEN plasmid integration into the P1 channel catfish genome. Neither the P1 nor the F1 mutant fish showed any noticeable changes in in body weight, survival rate, and hatching rate when the reproductive gene was knocked out. F1 families had a mean inheritance rate of 50.3%. The results brought us one step closer to allowing implementation of certain genetic techniques to aquaculture and fisheries management, while essentially eliminating the potential environment risk posed by transgenic, hybrid, and exotic fish as well as domestic fish.

The Application of Genetic and Genomic Biotechnology in Aquaculture.

This Special Issue, "The Application of Genetic and Genomic Biotechnology in Aquaculture," collates 14 published manuscripts covering different aspects of implementing advanced molecular genetics and genomic science in aquaculture [...].

Functionality of dietary antimicrobial peptides in aquatic animal health: Multiple meta-analyses.

Effects of antimicrobial peptides (AMP) added to diets on aquatic animal health and body function are influenced by multiple factors such as animal species, initial body weight, the dosage of AMP and feeding duration. However, there is limited knowledge on the relationship between these factors and the body function of aquatic animals. Here, we aimed to perform multiple meta-analyses to investigate the effects of dietary AMP on growth performance (feed conversion ratio [FCR], specific growth rate [SGR]), enzyme activity (superoxide dismutase activity [SOD], lysozyme activity [LSA]), disease resistance (cumulative survival rate [CSR], the expression of immune-related genes [GENE]) and the abundance of gut microbiota (MICRO) from a pool of empirical studies. Additionally, the dose-effect model was applied to determine the optimal AMP dose, initial body weight and feeding duration to maximize body function. To conduct the meta-analyses, we included 34 publications that estimated 705 effect sizes across 21 fish, 2 shrimp and 2 shellfish species. The results confirmed that the inclusion of AMP in the diet can significantly improve SGR, SOD, LSA, CSR and GENE and decrease FCR for aquatic animals. Interestingly, our findings implied a slight positive effect of AMP on MICRO albeit with a limited number of studies available on fish gut microbial communities. Although no significant linear or quadratic relationship was predicted by meta-regression, the dose-effect indicated that the optimal AMP doses for FCR, SGR, SOD and LSA were 707.5, 750.0, 1,050.0 and 937.5 mg/kg, respectively. Taken together, fish with an initial body weight of 30 g could be fed with a dose of 600 to 800 mg/kg for 2 mo when AMP-supplemented diets were applied in aquaculture, which can effectively improve body function and health while lowering aquafeed costs. In addition, more studies should address fish gut microbiota to delimitate the influence of dietary AMP on MICRO in the future.

Improved Growth and High Inheritance of Melanocortin-4 Receptor (mc4r) Mutation in CRISPR/Cas-9 Gene-Edited Channel Catfish, Ictalurus punctatus.

Effects of CRISPR/Cas9 knockout of the melanocortin-4 receptor (mc4r) gene in channel catfish, Ictalurus punctatus, were investigated. Three sgRNAs targeting the channel catfish mc4r gene in conjunction with Cas9 protein were microinjected in embryos and mutation rate, inheritance, and growth were studied. Efficient mutagenesis was achieved as demonstrated by PCR, Surveyor® assay, and DNA sequencing. An overall mutation rate of 33% and 33% homozygosity/bi-allelism was achieved in 2017. Approximately 71% of progeny inherited the mutation. Growth was generally higher in MC4R mutants than controls (CNTRL) at all life stages and in both pond and tank environments. There was a positive relationship between zygosity and growth, with F1 homozygous/bi-allelic mutants reaching market size 30% faster than F1 heterozygotes in earthen ponds (p = 0.022). At the stocker stage (~ 50 g), MC4R × MC4R mutants generated in 2019 were 40% larger than the mean of combined CNTRL × CNTRL families (p = 0.005) and 54% larger than F1 MC4R × CNTRL mutants (p = 0.001) indicating mutation may be recessive. With a high mutation rate and inheritance of the mutation as well as improved growth, the use of gene-edited MC4R channel catfish appears to be beneficial for application on commercial farms.

Transcriptome Analysis Reveals Key Gene Expression Changes in Blue Catfish Sperm in Response to Cryopreservation.

The hybrids of female channel catfish (Ictalurus punctatus) and male blue catfish (I. furcatus) account for >50% of US catfish production due to superior growth, feed conversion, and disease resistance compared to both parental species. However, these hybrids can rarely be naturally spawned. Sperm collection is a lethal procedure, and sperm samples are now cryopreserved for fertilization needs. Previous studies showed that variation in sperm quality causes variable embryo hatch rates, which is the limiting factor in hybrid catfish breeding. Biomarkers as indicators for sperm quality and reproductive success are currently lacking. To address this, we investigated expression changes caused by cryopreservation using transcriptome profiles of fresh and cryopreserved sperm. Sperm quality measurements revealed that cryopreservation significantly increased oxidative stress levels and DNA fragmentation, and reduced sperm kinematic parameters. The present RNA-seq study identified 849 upregulated genes after cryopreservation, including members of all five complexes in the mitochondrial electron transport chain, suggesting a boost in oxidative phosphorylation activities, which often lead to excessive production of reactive oxygen species (ROS) associated with cell death. Interestingly, functional enrichment analyses revealed compensatory changes in gene expression after cryopreservation to offset detrimental effects of ultra-cold storage: MnSOD was induced to control ROS production; chaperones and ubiquitin ligases were upregulated to correct misfolded proteins or direct them to degradation; negative regulators of apoptosis, amide biosynthesis, and cilium-related functions were also enriched. Our study provides insight into underlying molecular mechanisms of sperm cryoinjury and lays a foundation to further explore molecular biomarkers on cryo-survival and gamete quality.

Chromosome-level assembly and annotation of the blue catfish Ictalurus furcatus, an aquaculture species for hybrid catfish reproduction, epigenetics, and heterosis studies.

BACKGROUND: The blue catfish is of great value in aquaculture and recreational fisheries. The F1 hybrids of female channel catfish (Ictalurus punctatus) × male blue catfish (Ictalurusfurcatus) have been the primary driver of US catfish production in recent years because of superior growth, survival, and carcass yield. The channel-blue hybrid also provides an excellent model to investigate molecular mechanisms of environment-dependent heterosis. However, transcriptome and methylome studies suffered from low alignment rates to the channel catfish genome due to divergence, and the genome resources for blue catfish are not publicly available. RESULTS: The blue catfish genome assembly is 841.86 Mbp in length with excellent continuity (8.6 Mbp contig N50, 28.2 Mbp scaffold N50) and completeness (98.6% Eukaryota and 97.0% Actinopterygii BUSCO). A total of 30,971 protein-coding genes were predicted, of which 21,781 were supported by RNA sequencing evidence. Phylogenomic analyses revealed that it diverged from channel catfish approximately 9 million years ago with 15.7 million fixed nucleotide differences. The within-species single-nucleotide polymorphism (SNP) density is 0.32% between the most aquaculturally important blue catfish strains (D&B and Rio Grande). Gene family analysis discovered significant expansion of immune-related families in the blue catfish lineage, which may contribute to disease resistance in blue catfish. CONCLUSIONS: We reported the first high-quality, chromosome-level assembly of the blue catfish genome, which provides the necessary genomic tool kit for transcriptome and methylome analysis, SNP discovery and marker-assisted selection, gene editing and genome engineering, and reproductive enhancement of the blue catfish and hybrid catfish.

Response of cecropin transgenesis to challenge with Edwardsiella ictaluri in channel catfish Ictalurus punctatus.

Constructs bearing the cecropin B gene from the moth Hyalophora cecropia, driven by the cytomegalovirus (CMV) promoter, or the common carp beta-actin (ß-actin) promoter were transferred to channel catfish, Ictalurus punctatus via electroporation. One F3 channel catfish family transgenic for cecropin transgene driven by the CMV promoter, and one F1 channel catfish family transgenic for cecropin transgene driven by the common carp ß-actin promoter were produced. F3 and F1 individuals exhibited enhanced disease resistance when challenged in tanks with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC). Inheritance of the transgene by the F1 and F3 generation was 15% and 60%, respectively. Growth rates of the cecropin transgenic and non-transgenic full siblings (controls) channel catfish were not different (P > 0.05). All transgenic fish showed significant resistance to infection by ESC at day 3 and day 4 post exposure (P = 0.005). No correlation was detected between body weight and time to death for all genetic groups (P = 0.34). Results of our study confirmed that genetic enhancement of E. ictaluri resistance can be achieved by cecropin transgenesis in channel catfish. In addition to survival rate, improving survival time is essential because the extension of survival time gives a better chance to apply treatments to stop the bacterial infection.

Gene Editing of the Catfish Gonadotropin-Releasing Hormone Gene and Hormone Therapy to Control the Reproduction in Channel Catfish, Ictalurus punctatus.

Transcription activator-like effector nuclease (TALEN) plasmids targeting the channel catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). Targeted cfGnRH fish were sequenced and base deletion, substitution, and insertion were detected. The gene mutagenesis was achieved in 52.9% of P1 fish. P1 mutants (individuals with human-induced sequence changes at the cfGnRH locus) had lower spawning rates (20.0−50.0%) when there was no hormone therapy compared to the control pairs (66.7%) as well as having lower average egg hatch rates (2.0% versus 32.3−74.3%) except for one cfGnRH mutated female that had a 66.0% hatch rate. After low fertility was observed in 2016, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants in 2017, which were not significantly different from the controls (p > 0.05). No exogenous DNA fragments were detected in the genome of mutant P1 fish, indicating no integration of the plasmids. No obvious effects on other economically important traits were observed after the knockout of the reproductive gene in the P1 fish. Growth rates, survival, and appearance between mutant and control individuals were not different. While complete knock-out of reproductive output was not achieved, as these were mosaic P1 brood stock, gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.

Identification of an Epigenetically Marked Locus within the Sex Determination Region of Channel Catfish.

Channel catfish has an XY sex determination system. However, the X and Y chromosomes harbor an identical gene content of 950 genes each. In this study, we conducted comparative analyses of methylome and transcriptome of genetic males and genetic females before gonadal differentiation to provide insights into the mechanisms of sex determination. Differentially methylated CpG sites (DMCs) were predominantly identified on the sex chromosome, most notably within the sex determination region (SDR), although the overall methylation profiles across the entire genome were similar between genetic males and females. The drastic differences in methylation were located within the SDR at nucleotide position 14.0-20.3 Mb of the sex chromosome, making this region an epigenetically marked locus within the sex determination region. Most of the differentially methylated CpG sites were hypermethylated in females and hypomethylated in males, suggesting potential involvement of methylation modification in sex determination in channel catfish. Along with the differential methylation in the SDR, a number of differentially expressed genes within the SDR were also identified between genetic males and females, making them potential candidate genes for sex determination and differentiation in channel catfish.

Environment-Dependent Heterosis and Transgressive Gene Expression in Reciprocal Hybrids between the Channel Catfish Ictalurus punctatus and the Blue Catfish Ictalurus furcatus.

The hybrid between female channel catfish (Ictalurus punctatus) and male blue catfish (Ictalurus furcatus) is superior in feed conversion, disease resistance, carcass yield, and harvestability compared to both parental species. However, heterosis and heterobeltiosis only occur in pond culture, and channel catfish grow much faster than the other genetic types in small culture units. This environment-dependent heterosis is intriguing, but the underlying genetic mechanisms are not well understood. In this study, phenotypic characterization and transcriptomic analyses were performed in the channel catfish, blue catfish, and their reciprocal F1s reared in tanks. The results showed that the channel catfish is superior in growth-related morphometrics, presumably due to significantly lower innate immune function, as investigated by reduced lysozyme activity and alternative complement activity. RNA-seq analysis revealed that genes involved in fatty acid metabolism/transport are significantly upregulated in channel catfish compared to blue catfish and hybrids, which also contributes to the growth phenotype. Interestingly, hybrids have a 40-80% elevation in blood glucose than the parental species, which can be explained by a phenomenon called transgressive expression (overexpression/underexpression in F1s than the parental species). A total of 1140 transgressive genes were identified in F1 hybrids, indicating that 8.5% of the transcriptome displayed transgressive expression. Transgressive genes upregulated in F1s are enriched for glycan degradation function, directly related to the increase in blood glucose level. This study is the first to explore molecular mechanisms of environment-dependent heterosis/heterobeltiosis in a vertebrate species and sheds light on the regulation and evolution of heterosis vs. hybrid incompatibility.

Comparison of growth performance among channel-blue hybrid catfish, ccGH transgenic channel catfish, and channel catfish in a tank culture system.

Fish is an essential source of high-quality protein for people worldwide. The present study was designed to compare the growth performance among the channel-blue hybrid catfish, channel catfish transgenic for the channel catfish growth hormone (ccGH) cDNA driven by the antifreeze protein promoter from an ocean pout Zoarces americanus (opAFP-ccGH), and non-transgenic channel catfish control. Mean body weight of channel-blue hybrid catfish was 15.80 and 24.06% larger than non-transgenic channel catfish control at 4 and 18 months of age, respectively. However, transgenic opAFP-ccGH channel catfish were 5.52 and 43.41% larger than channel-blue hybrid catfish and 22.19 and 77.91% larger than their controls at 4 and 18 months of age, respectively. Significant differences in mean body weight between the sexes within all genetic types were found. Males were larger than females (P < 0.001). However, mean body weight of non-transgenic males was not larger than transgenic opAFP-ccGH females or male and female hybrid catfish. Condition factor of transgenic opAFP-ccGH channel catfish was higher (P < 0.05) than that of full-sibling, non-transgenic channel catfish and hybrid catfish. The mean percentage body weight gain of GH transgenic channel catfish was 559%, the channel-blue hybrid catfish was 384.9% and their non-transgenic controls channel catfish was 352.6%.

Growth Differences of Growth Hormone Transgenic Female and Male Channel Catfish, Ictalurus punctatus, Grown in Earthen Ponds to Sexual Maturation.

This study compared growth performance between female and male transgenic channel catfish, Ictalurus punctatus, containing channel catfish growth hormone full-length cDNA driven by the ocean pout antifreeze protein promoter, opAFP-ccGH, the rainbow trout metallothionein promoter, rtMT-ccGH, or both constructs, and their non-transgenic siblings in earthen ponds at 16 and 48 months of age. Body weight between the transgenic and their non-transgenic siblings differed (P < 0.001) at all ages. Transgenic F2 opAFP-ccGH grew 1.51- to 2.58-, F2 rtMT-ccGH grew 1.44- to 2.99- and F1fish transgenic for both constructs grew 1.36- to 2.92- fold larger than their non-transgenic sibling controls, depending upon age and sex. Body weight of the transgenic GH males was significantly higher than those of the transgenic GH females at 16 months of age (P < 0.001). However, body weight of the transgenic GH females was significantly higher (P < 0.001) compared with those of the transgenic GH males at 48 months of age, but not for the double transgenics (P > 0.05). In the case of non-transgenic GH siblings, males were larger than females at both 16 and 48 months of age (P < 0.001). Sexually dimorphic responses to GH transgenes were the opposite after sexual maturation. When critically low dissolved oxygen levels were encountered, survival of transgenic male and female opAFP-ccGH channel catfish was lower than that of controls (P = 0.004), as well as rtMT-ccGH females (P = 0.11), which is not surprising since the largest fish are most likely to succumb during an oxygen depletion.

Effectiveness of Cathelicidin Antimicrobial Peptide against Ictalurid Catfish Bacterial Pathogens.

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 × 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 µg 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.

Direct and pleiotropic effects of the Masou Salmon Delta-5 Desaturase transgene in F1 channel catfish (Ictalurus punctatus).

Channel catfish (Ictalurus punctatus) is the primary culture species in the US along with its hybrid made with male blue catfish, I. furcatus. In an effort to improve the nutritional value of channel catfish, the masou salmon Δ5-desaturase like gene (D5D) driven by the common carp beta-actin promoter (ßactin) was inserted into channel catfish. The objectives of this study were to determine the effectiveness of ßactin-D5D for improving n-3 fatty acid production in F1 transgenic channel catfish, as well as examine pleiotropic effects on growth, proximate analysis, disease resistance, and other performance traits. Transgenic F1 channel catfish showed a 33% increase in the relative proportion of n-3 fatty acids coupled with a 15% decrease in n-6 fatty acids and a 17% decrease in n-9 fatty acids when compared to non-transgenic full-siblings (P < 0.01, P < 0.01, P < 0.01). However, while the relative proportion of n-3 fatty acids was achieved, the total amount of fatty acids in the transgenic fish decreased resulting in a reduction of all fatty acids. Insertion of the ßactin-D5D transgene into channel catfish also had large effects on the body composition, and growth of channel catfish. Transgenic channel catfish grew faster, were more disease resistant, had higher protein and moisture percentage, but lower fat percentage than full-sib controls. There were sex effects as performance changes were more dramatic and significant in males. The ßactin-D5D transgenic channel catfish were also more uniform in their fatty acid composition, growth and other traits.

Comparative Genomic and Transcriptomic Analyses Revealed Twenty-Six Candidate Genes Involved in the Air-Breathing Development and Function of the Bighead Catfish Clarias macrocephalus.

The bighead catfish (Clarias macrocephalus) and channel catfish (Ictalurus punctatus) are freshwater species in the Siluriformes order. C. macrocephalus has both gills and modified gill structures serving as an air-breathing organ (ABO), while I. punctatus does not possess such an organ, and cannot breathe in air, providing an excellent model for studying the molecular basis of ABO development in teleost fish. To investigate the critical time window for the development of air-breathing function, seven development stages were selected based on hypoxia challenge results, and RNA-seq was performed upon C. macrocephalus to compare with the non-air-breathing I. punctatus. Five-hundred million reads were generated and 25,239 expressed genes were annotated in C. macrocephalus. Among those, 8675 genes were differentially expressed across developmental stages. Comparative genomic analysis identified 1458 C. macrocephalus specific genes, which were absent in I. punctatus. Gene network and protein-protein interaction analyses identified 26 key hub genes involved in the air-breathing function. Three top candidate genes, mb, ngb, hbae, are mainly associated with oxygen carrying, oxygen binding, and heme binding activities. Our study provides a rich data set for exploring the genomic basis of air-breathing function in C. macrocephalus and offers insights into the adaption to hypoxic environments.

CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome.

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 ß-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.

Development of a spermatogonia cryopreservation protocol for blue catfish, Ictalurus furcatus.

Sustainability of channel catfish, Ictalurus punctatus ♀ × blue catfish, Ictalurus furcatus ♂ hybrid aquaculture relies on new innovative technologies to maximize fry output. Transplanting spermatogonial stem cells (SSCs) from blue catfish into channel catfish hosts has the potential to greatly increase gamete availability and improve hybrid catfish fry outputs. Cryopreservation would make these cells readily accessible for xenogenesis, but a freezing protocol for blue catfish testicular tissues has not yet been fully developed. Therefore, the objectives of this experiment were to identify the best permeating [dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol, methanol] and non-permeating (lactose or trehalose with egg yolk or BSA) cryoprotectants, their optimal concentrations, and the best freezing rates (-0.5, -1.0, -5.0, -10 °C/min until -80 °C) that yield the highest number of viable type A spermatogonia cells. Results showed that all of these factors had significant impacts on post-thaw cell production and viability. DMSO was the most efficient permeating cryoprotectant at a concentration of 1.0 M. The optimal concentration of each cryoprotectant depended on the specific cryoprotectant due to interactions between the two factors. Of the non-permeating cryoprotectants, 0.2 M lactose with egg yolk consistently improved type A spermatogonia production and viability beyond that of the 1.0 M DMSO control. The overall best freezing rate was consistent at -1 °C/min, but similar results were obtained using -0.5 °C/min. Overall, we recommend cryopreserving blue catfish testicular tissues in 1.0 M DMSO with 0.2 M lactose and egg yolk at a rate of either -0.5 or -1 °C/min to achieve the best cryopreservation outcomes. Continued development of cryopreservation protocols for blue catfish and other species will make spermatogonia available for xenogenic applications and genetic improvement programs.

Cathelicidins enhance protection of channel catfish, Ictalurus punctatus, and channel catfish ♀ × blue catfish, Ictalurus furcatus ♂ hybrid catfish against Edwarsiella ictaluri infection.

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.