Reversible Sterilization of Channel Catfish via Overexpression of Glutamic Acid Decarboxylase Gene.

The confinement of transgenic fish is essential to prevent their escape and reproduction in natural ecosystems. Reversible transgenic sterilization is a promising approach to control the reproduction of transgenic fish. Therefore, the present study was conducted to develop a reversibly sterile channel catfish (Ictalurus punctatus) via the transgenic overexpression of the goldfish (Carassius auratus) glutamic acid decarboxylase (GAD) gene driven by the common carp (Cyprinus carpio) ß-actin promoter to disrupt normal gamma-aminobutyric acid (GABA) regulation. Three generations of GAD-transgenic fish were produced. All studied generations showed repressed reproductive performance; however, this was not always statistically significant. In F1, 5.4% of the transgenic fish showed a sexual maturity score ≥ 4 (maximum = 5) at five years of age, which was lower (p = 0.07) than that of the control group (16.8%). In the spawning experiments conducted on F1 transgenic fish at six and nine years of age, 45.5% and 20.0% of fish spawned naturally, representing lower values (p = 0.09 and 0.12, respectively) than the percentages in the sibling control fish of the same age (83.3% and 66.7%, respectively). Four of six pairs of the putative infertile six-year-old fish spawned successfully after luteinizing hormone-releasing hormone analog (LHRHa) therapy. Similar outcomes were noted in the three-year-old F2 fish, with a lower spawning percentage in transgenic fish (20.0%) than in the control (66.7%). In one-year-old F2-generation transgenic fish, the observed mean serum gonadotropin-releasing hormone (GnRH) levels were 9.23 ± 2.49 and 8.14 ± 2.21 ng/mL for the females and males, respectively. In the control fish, the mean levels of GnRH were 11.04 ± 4.06 and 9.03 ± 2.36 ng/mL for the females and males, respectively, which did not differ significantly from the control (p = 0.15 and 0.27 for females and males, respectively). There was no significant difference in the estradiol levels of the female transgenic and non-transgenic fish in the one- and four-year-old F2-generation fish. The four-year-old F2-generation male transgenic fish exhibited significantly (p < 0.05) lower levels of GnRH and testosterone than the control fish. In conclusion, while overexpressing GAD repressed the reproductive abilities of channel catfish, it did not completely sterilize transgenic fish. The sterilization rate might be improved through selection in future generations.

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.

Fine Mapping of QTLs for Alkaline Tolerance in Crucian Carp (Carassius auratus) Using Genome-Wide SNP Markers.

Crucian carp (Carassius auratus) is widely distributed in the world and has become an economically freshwater fish. The population in Lake Dali Nur can tolerate the extreme alkaline environment with alkalinity over 50 mmol/L (pH 9.6), thus providing a special model for exploring alkali-tolerant molecular markers in an extremely alkaline environment. In this study, we constructed a high-density and high-resolution linkage map with 16,224 SNP markers based on genotyping-by-sequencing (GBS) consisting of 152 progenies and conducted QTL studies for alkali-tolerant traits. The total length of the linkage map was 3918.893 cM, with an average distance of 0.241 cM. Two QTLs for the ammonia-N-tolerant trait were detected on LG27 and LG45. A QTL for the urea-N-tolerant trait was detected on LG27. Interestingly, mapping the two QTLs on LG27 revealed that the mapped genes were both located in the intron of CDC42. GO functional annotation and KEGG enrichment analysis results indicated that the biological functions might be involved in the cell cycle, cellular senescence, MAPK, and Ras signaling pathways. These findings suggest that CDC42 may play an important role in the process of dealing with extremely alkaline environments.

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.

Using UAV-Based Temporal Spectral Indices to Dissect Changes in the Stay-Green Trait in Wheat.

Stay-green (SG) in wheat is a beneficial trait that increases yield and stress tolerance. However, conventional phenotyping techniques limited the understanding of its genetic basis. Spectral indices (SIs) as non-destructive tools to evaluate crop temporal senescence provide an alternative strategy. Here, we applied SIs to monitor the senescence dynamics of 565 diverse wheat accessions from anthesis to maturation stages over 2 field seasons. Four SIs (normalized difference vegetation index, green normalized difference vegetation index, normalized difference red edge index, and optimized soil-adjusted vegetation index) were normalized to develop relative stay-green scores (RSGS) as the SG indicators. An RSGS-based genome-wide association study identified 47 high-confidence quantitative trait loci (QTL) harboring 3,079 single-nucleotide polymorphisms associated with SG and 1,085 corresponding candidate genes. Among them, 15 QTL overlapped or were adjacent to known SG-related QTL/genes, while the remaining QTL were novel. Notably, a set of favorable haplotypes of SG-related candidate genes such as TraesCS2A03G1081100, TracesCS6B03G0356400, and TracesCS2B03G1299500 are increasing following the Green Revolution, further validating the feasibility of the pipeline. This study provided a valuable reference for further quantitative SG and genetic research in diverse wheat panels.

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.

Slow stripe rusting in Chinese wheat Jimai 44 conferred by Yr29 in combination with a major QTL on chromosome arm 6AL.

KEY MESSAGE: YrJ44, a more effective slow rusting gene than Yr29, was localized to a 3.5-cM interval between AQP markers AX-109373479 and AX-109563479 on chromosome 6AL. "Slow rusting" (SR) is a type of adult plant resistance (APR) that can provide non-specific durable resistance to stripe rust in wheat. Chinese elite wheat cultivar Jimai 44 (JM44) has maintained SR to stripe rust in China since its release despite exposure to a changing and variable pathogen population. An F2:6 population comprising 295 recombinant inbred lines (RILs) derived from a cross between JM44 and susceptible cultivar Jimai 229 (JM229) was used in genetic analysis of the SR. The RILs and parental lines were evaluated for stripe rust response in five field environments and genotyped using the Affymetrix Wheat55K SNP array and 13 allele-specific quantitative PCR-based (AQP) markers. Two stable QTL on chromosome arms 1BL and 6AL were identified by inclusive composite interval mapping. The 1BL QTL was probably the pleiotropic gene Lr46/Yr29/Sr58. QYr.nwafu-6AL (hereafter named YrJ44), mapped in a 3.5-cM interval between AQP markers AX-109373479 and AX-109563479, was more effective than Yr29 in reducing disease severity and relative area under the disease progress curve (rAUDPC). RILs harboring both YrJ44 and Yr29 displayed levels of SR equal to the resistant parent JM44. The AQP markers linked with YrJ44 were polymorphic and significantly correlated with stripe rust resistance in a panel of 1,019 wheat cultivars and breeding lines. These results suggested that adequate SR resistance can be obtained by combining YrJ44 and Yr29 and the AQP markers can be used in breeding for durable stripe rust resistance.

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.

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.

Gene-Based Genome-Wide Association Study Identified Genes for Agronomic Traits in Maize.

A gene integrates the effects of all SNPs in its sequence span, which benefits the genome-wide association study. To explore gene-level variations affecting economic traits in maize, we extended the SNP-based GWAS analysis software Single-RunKing developed by our team to gene-based GWAS, which used the FaST-LMM algorithm to convert the linear mixed model into simple linear model association analysis. An F-test statistic was formulated to test and identify candidate genes. We compared the statistical efficiency of using 80% principal components (EPC), the first principal component (FPC), and all SNP markers (ALLSNP) as independent variables, which predecessors commonly used to integrate SNPs and represent genes. With a Huazhong Agricultural University (HAU) genomic dataset of 2.65M SNPs from 540 maize plants, 34,774 genes were annotated across the whole genome. Genome-wide association studies with 20 agronomic traits were performed using the software developed here. Another maize dataset from the Ames panel (AP) was also analyzed. The EPC method fits the model well and has good statistical efficiency. It not only overcomes the false negative problem when using all SNP markers for analysis (ALLSNP) but also solves the false positive problem of its corresponding simple linear model method EPCLM. Compared with FPC, the EPC method has higher statistical efficiency. A total of 132 quantitative trait genes (QTG) were identified for the 20 traits from HAU maize dataset and one trait of AP maize.

Fusing attention mechanism with Mask R-CNN for instance segmentation of grape cluster in the field.

Accurately detecting and segmenting grape cluster in the field is fundamental for precision viticulture. In this paper, a new backbone network, ResNet50-FPN-ED, was proposed to improve Mask R-CNN instance segmentation so that the detection and segmentation performance can be improved under complex environments, cluster shape variations, leaf shading, trunk occlusion, and grapes overlapping. An Efficient Channel Attention (ECA) mechanism was first introduced in the backbone network to correct the extracted features for better grape cluster detection. To obtain detailed feature map information, Dense Upsampling Convolution (DUC) was used in feature pyramid fusion to improve model segmentation accuracy. Moreover, model generalization performance was also improved by training the model on two different datasets. The developed algorithm was validated on a large dataset with 682 annotated images, where the experimental results indicate that the model achieves an Average Precision (AP) of 60.1% on object detection and 59.5% on instance segmentation. Particularly, on object detection task, the AP improved by 1.4% and 1.8% over the original Mask R-CNN (ResNet50-FPN) and Faster R-CNN (ResNet50-FPN). For the instance segmentation, the AP improved by 1.6% and 2.2% over the original Mask R-CNN and SOLOv2. When tested on different datasets, the improved model had high detection and segmentation accuracy and inter-varietal generalization performance in complex growth environments, which is able to provide technical support for intelligent vineyard management.

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.

Traffic Safety Improvement via Optimizing Light Environment in Highway Tunnels.

Driving in tunnel areas depends more heavily on light conditions than that on open roadways. Traditional lighting systems in highway tunnels adjust lighting parameters only caring about outside light luminance, and focus is usually on energy conservation; however, little concern is about drivers' actual physical and psychological needs. How to leverage the enormous research progress of traffic safety, light environment, human factors engineering, and modern lighting sources to create an ideal tunnel light environment that aids with ensuring driving safety and lower interference effects caused by the change of light environment will greatly improve safety level and reduce adverse influence on drivers' visual health in a tunnel area. An intelligent lighting control system designed with multiple influence factors are systematically considered. Based on sensor data from outside natural light conditions, target lighting parameters are determined per each lighting zone requires; then, lighting commands will be transferred and parsed by adaptive lighting controllers and modules, eventually LED lighting properties are altered step by step. This system helps a lot with optimizing tunnel lighting quality and improving drivers' visual performance; as a result, it contributes to lower the fluctuation of drivers' workload and get a smooth traffic flow, and ultimately this technically ensures physical and mental health of drivers in a tunnel area.

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.