Advancing aquaculture: Production of xenogenic catfish by transplanting blue catfish (Ictalurus furcatus) and channel catfish (I. punctatus) stem cells into white catfish (Ameiurus catus) triploid fry.

Xenogenesis has been recognized as a prospective method for producing channel catfish, Ictalurus punctatus ♀ × blue catfish, I. furcatus ♂ hybrids. The xenogenesis procedure can be achieved by transplanting undifferentiated stem cells derived from a donor fish into a sterile recipient. Xenogenesis for hybrid catfish embryo production has been accomplished using triploid channel catfish as a surrogate. However, having a surrogate species with a shorter maturation period, like white catfish (Ameiurus catus), would result in reduced feed costs, labor costs, and smaller body size requirements, making it a more suitable species for commercial applications where space is limited, and as a model species. Hence, the present study was conducted to assess the effectiveness of triploid white catfish as a surrogate species to transplant blue catfish stem cells (BSCs) and channel catfish stem cells (CSCs). Triploid white catfish fry were injected with either BSCs or CSCs labeled with PKH 26 fluorescence dye from 0 to 12 days post hatch (DPH). No significant differences in weight and length of fry were detected among BSCs and CSCs injection times (0 to 12 DPH) when fry were sampled at 45 and 90 DPH (P > 0.05). The highest survival was reported when fry were injected between 4.0 to 5.5 DPH (≥ 81.2%). At 45 and 90 DPH, cell and cluster area increased for recipients injected from 0 to 5.2 DPH, and the highest cluster area values were reported between 4.0 to 5.2 DPH. Thereafter, fluorescent cell and cluster area in the host declined with no further decrease after 10 DPH. At 45 DPH, the highest percentage of xenogens were detected when fry were injected with BSCs between 4.0 to 5.0 and CSCs between 3.0 to 5.0 DPH. At 90 DPH, the highest number of xenogens were detected from 4.0 to 6.0 DPH when injected with either BSCs or CSCs. The current study demonstrated the suitability of white catfish as a surrogate species when BSCs and CSCs were transplanted into triploid white catfish between 4.0 to 6.0 DPH (27.4 ± 0.4°C). Overall, these findings allow enhanced efficiency of commercializing xenogenic catfish carrying gametes of either blue catfish or channel catfish.

Transcriptome analysis revealed potential mechanisms of channel catfish growth advantage over blue catfish in a tank culture environment.

Channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus) are two economically important freshwater aquaculture species in the United States, with channel catfish contributing to nearly half of the country's aquaculture production. While differences in economic traits such as growth rate and disease resistance have been noted, the extent of transcriptomic variance across various tissues between these species remains largely unexplored. The hybridization of female channel catfish with male blue catfish has led to the development of superior hybrid catfish breeds that exhibit enhanced growth rates and improved disease resistance, which dominate more than half of the total US catfish production. While hybrid catfish have significant growth advantages in earthen ponds, channel catfish were reported to grow faster in tank culture environments. In this study, we confirmed channel fish's superiority in growth over blue catfish in 60-L tanks at 10.8 months of age (30.3 g and 11.6 g in this study, respectively; p < 0.001). In addition, we conducted RNA sequencing experiments and established transcriptomic resources for the heart, liver, intestine, mucus, and muscle of both species. The number of expressed genes varied across tissues, ranging from 5,036 in the muscle to over 20,000 in the mucus. Gene Ontology analysis has revealed the functional specificity of differentially expressed genes within their respective tissues, with significant pathway enrichment in metabolic pathways, immune activity, and stress responses. Noteworthy tissue-specific marker genes, including lrrc10, fabp2, myog, pth1a, hspa9, cyp21a2, agt, and ngtb, have been identified. This transcriptome resource is poised to support future investigations into the molecular mechanisms underlying environment-dependent heterosis and advance genetic breeding efforts of hybrid catfish.

From heterosis to outbreeding depression: genotype-by-environment interaction shifts hybrid fitness in opposite directions.

In F1 hybrids, phenotypic values are expected to be near the parental means under additive effects or close to one parent under dominance. However, F1 traits can fall outside the parental range, and outbreeding depression occurs when inferior fitness is observed in hybrids. Another possible outcome is heterosis, a phenomenon that interspecific hybrids or intraspecific crossbred F1s exhibit improved fitness compared to both parental species or strains. As an application of heterosis, hybrids between channel catfish females and blue catfish males are superior in feed conversion efficiency, carcass yield, and harvestability. Over twenty years of hybrid catfish production in experimental settings and farming practices generated abundant phenotypic data, making it an ideal system to investigate heterosis. In this study, we characterized fitness in terms of growth and survival longitudinally, revealing environment-dependent heterosis. In ponds, hybrids outgrow both parents due to an extra rapid growth phase of 2∼4 months in year 2. This bimodal growth pattern is unique to F1 hybrids in pond culture environments only. In sharp contrast, the same genetic types cultured in tanks display outbreeding depression, where hybrids perform poorly, while channel catfish demonstrate superiority in growth throughout development. Our findings represent the first example, known to the authors, of opposite fitness shifts in response to environmental changes in interspecific vertebrate hybrids, suggesting a broader fitness landscape for F1 hybrids. Future genomic studies based on this experiment will help understand genome-environment interaction in shaping the F1 progeny fitness in the scenario of environment-dependent heterosis and outbreeding depression.

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.

Effect of dosage of orally administered 17α-methyltestosterone on sex reversion of the yellowtail tetra Astyanax lacustris (Lütken, 1875).

The females of yellowtail tetra (Astyanax lacustris), known as the freshwater sardine, are approximately 1.33 times larger than males, and thus, all-female monosex culture would increase production and reduce size variability. The present work aimed to identify the optimal dose of 17α-methyltestosterone (MT) to be used in the masculinization of A. lacustris for indirect sex reversal. Three different concentrations of MT (20, 40, and 60 mg/kg of feed in the diet) were fed to the fry for 30 days. Thirty adult individuals from each treatment, including the control (0 mg MT/kg), were evaluated for gonadal development, morphological and histological sexual identification, zootechnical performance, and the possible genotoxic effect caused by prolonged exposure to MT. MT significantly (P<0.01) affected the differentiation of the gonads, with the presence of possible inhibitory effects in all treatments. Intersex individuals were present in the 20 and 60 mg MT/kg treatments. All treatments were able to masculinize A. lacustris and the treatment with the lowest hormone concentration produced the highest percentage of males 76.7%, while the control had 46.7% males. The presence of erythrocyte nuclear alterations indicated a possible cytotoxic effect of MT in treatments 40 and 60 mg MT/kg, however, the use of the hormone did not affect the growth and the survival of the individuals. Thus, the use of MT is a viable option for obtaining neomales as a first step into the production of all-female progenies.

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.

Genomic imprinting-like monoallelic paternal expression determines sex of channel catfish.

The X and Y chromosomes of channel catfish have the same gene contents. Here, we report allelic hypermethylation of the X chromosome within the sex determination region (SDR). Accordingly, the X-borne hydin-1 gene was silenced, whereas the Y-borne hydin-1 gene was expressed, making monoallelic expression of hydin-1 responsible for sex determination, much like genomic imprinting. Treatment with a methylation inhibitor, 5-aza-dC, erased the epigenetic marks within the SDR and caused sex reversal of genetic females into phenotypic males. After the treatment, hydin-1 and six other genes related to cell cycle control and proliferative growth were up-regulated, while three genes related to female sex differentiation were down-regulated in genetic females, providing additional support for epigenetic sex determination in catfish. This mechanism of sex determination provides insights into the plasticity of genetic sex determination in lower vertebrates and its connection with temperature sex determination where DNA methylation is broadly involved.

A New Strategy for Increasing Knock-in Efficiency: Multiple Elongase and Desaturase Transgenes Knock-in by Targeting Long Repeated Sequences.

CRISPR/Cas9-mediated knock-in (KI) has a wide application in gene therapy, gene function study, and transgenic breeding programs. Unlike gene therapy, which requires accurate KI to correct gene mutation, transgenic breeding programs can accept robust KI as long as integration does not interrupt normal gene functions and result in any negative pleiotropic effects. High KI efficiency is required to reduce the breeding cost and shorten the breeding period, especially in transferring multiple foreign genes to a single individual. To elevate the KI efficacy and achieve multiple gene KIs simultaneously, we introduced a new strategy that enables transgene integration into numerous sites of the genome by targeting long repeated sequences (LRSs). Using this simple strategy, for the first time we successfully generated transgenic fish carrying the masu salmon (Oncorhynchus masou) elovl2 gene and rabbitfish (Siganus canaliculatus) Δ4 fad and Δ6 fad genes, and achieved robust target KI of elovl2 and Δ6 fad genes at multiple sites of LRS1 and LRS3, respectively, in the initial generation. This demonstrated that donor plasmid homology arms, which were nearly identical but not completely the same as the genome sequence, still led to on-target KI. Although the target KI efficiencies at LRS1, LRS2, and LRS3 sites were still relatively low in the current study, it is very promising that 100% KI efficiency in the future could be realized and perfected by selection of better LRSs and optimization of sgRNAs.

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.

Feminization of channel catfish with 17ß-oestradiol involves methylation and expression of a specific set of genes independent of the sex determination region.

Exogenous oestrogen 17ß-oestradiol (E2) has been shown to effectively induce feminization in teleosts. However, the molecular mechanisms underlying the process remain unclear. Here, we determined global DNA methylation and gene expression profiles of channel catfish (Ictalurus punctatus) during early sex differentiation after E2 treatment. Overall, the levels of global DNA methylation after E2 treatment were not significantly different from those of controls. However, a specific set of genes were differentially methylated, which included many sex differentiation-related pathways, such as MARK signalling, adrenergic signalling, Wnt signalling, GnRH signalling, ErbB signalling, and ECM-receptor interactions. Many genes involved in these pathways were also differentially expressed after E2 treatment. Specifically, E2 treatments resulted in upregulation of female-related genes and downregulation of male-related genes in genetic males during sex reversal. However, E2-induced sex reversal did not cause sex-specific changes in methylation profiles or gene expression within the sex determination region (SDR) on chromosome 4, suggesting that E2-induced sex reversal was a downstream process independent of the sex determination process that was regulated by sex-specific methylation within the SDR.

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.

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.

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.

CRISPR/Cas9-Mediated Transgenesis of the Masu Salmon (Oncorhynchus masou) elovl2 Gene Improves n-3 Fatty Acid Content in Channel Catfish (Ictalurus punctatus).

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), particularly eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), play a very important role in human health. Channel catfish (Ictalurus punctatus) is one of the leading freshwater aquaculture species in the USA, but has low levels of EPA and DHA compared to some fish such as salmon. To improve EPA and DHA content, a modification of the n-3 PUFA biosynthetic pathway was achieved through the insertion of an elovl2 transgene isolated from masu salmon (Oncorhynchus masou) driven by a carp ß-actin promoter using a two-hit by gRNA and two oligos with a targeting plasmid (2H2OP) CRISPR/Cas9 approach. Integration rate of the transgene was high (37.5%) and detected in twelve different tissues of P1 transgenic fish with tissue-specific gene expression. Liver and muscle had relative high gene expression (13.4- and 9.2-fold change, respectively). Fatty acid analysis showed DHA content in the muscle from transgenic fish was 1.62-fold higher than in non-transgenic fish (P < 0.05). Additionally, total n-3 PUFAs and omega-6 polyunsaturated fatty acids (n-6 PUFAs) increased to 1.41-fold and 1.50-fold, respectively, suggesting the ß-actin-elovl2 transgene improved biosynthesis of PUFAs in channel catfish as a whole. The n-9 fatty acid level decreased in the transgenic fish compared to the control. Morphometric analysis showed that there were significant differences between injected fish with sgRNAs (including positive and negative fish) and sham-injected controls (P < 0.001). Potential off-target effects are likely the major factor responsible for morphological deformities. Optimization of sgRNA design to maximize activity and reduce off-target effects of CRISPR/Cas9 should be examined in future transgenic research, but this research shows a promising first step in the improvement of n-3 PUFAs in channel catfish.

Allelically and Differentially Expressed Genes After Infection of Edwardsiella ictaluri in Channel Catfish as Determined by Bulk Segregant RNA-Seq.

Identification of genetic markers associated with resistance against enteric septicemia of catfish (ESC) is of great interest for genetic enhancement programs of catfish. In the present study, bulk segregant RNA-Seq analysis was applied to determine differentially expressed genes and alleles after ESC infection. Here we report three genomic regions on LG1, LG12, and LG26, containing significant single-nucleotide polymorphisms (SNPs). These genomic regions aligned well with quantitative trait loci (QTL) previously identified. Within the QTL regions, eleven genes were found to be differentially regulated between phenotypic bulks. Importantly, the QTL on linkage group 1 (LG1) were found to be expressed in the liver, whereas the QTL on LG12 and LG26 were expressed in the intestine, suggesting multiple mechanisms of ESC resistance. It is apparent that apolipoproteins may be important for ESC resistance as the QTL on LG1 included the 14-kDa apolipoprotein genes that are both allelically expressed and differentially expressed between the resistant and susceptible bulks. Traf2 and NCK-interacting protein kinase (TNIK) were found in the QTL on LG12, and it was downregulated in resistant fish, suggesting the importance of NCK downregulation in ESC resistance, as previously reported. In addition, we observed divergent gene expression patterns between the liver and intestine after infection. Immune/inflammatory-related processes were overrepresented from liver DEGs, while those DEGs identified from intestine were enriched for proteolysis and wounding processes. Taken together, the BSR-Seq analysis presented here advanced the knowledge of ESC resistance, providing information of not only positions of QTL but also genes and their differential expression between resistant and susceptible fish, making it one step closer to the identification of the causal genes for ESC resistance.