Synergistic Effect of Dietary Supplementation with Sodium Butyrate, ß-Glucan and Vitamins on Growth Performance, Cortisol Level, Intestinal Microbiome and Expression of Immune-Related Genes in Juvenile African Catfish (Clarias gariepinus).

The effect of dietary supplementation with sodium butyrate, ß-glucan and vitamins (A, D3, E, K, C) on breeding indicators and immune parameters of juvenile African catfish was examined. The fish were fed with unenriched (group C) and enriched feed with a variable proportion of sodium butyrate/ß-glucan, and constant content of vitamins (W1-W3). After the experiment, blood and the middle gut were collected. The microbiome of the gut was determined using Next Generation Sequencing (NGS). Liver tissue was collected for determination of expression of immune-related genes (HSP70, IL-1ß, TNFα). W2 and W3 were characterized by the most favorable values of breeding indicators (p < 0.05). The highest blood cortisol concentration was in group C (71.25 ± 10.45 ng/mL), and significantly the lowest in W1 (46.03 ± 7.01 ng/ mL) (p < 0.05). The dominance of Cetobacterium was observed in all study groups, with the largest share in W3 (65.25%) and W1 (61.44%). Gene expression showed an increased number of HSP70 genes in W1. IL-1ß and TNFα genes peaked at W3. The W3 variant turns out to be the most beneficial supplementation, due to the improvement of breeding and immunological parameters. The data obtained can be used to create a preparation for commercial use in the breeding of this species.

Complete mitochondrial genome of critically endangered catfish Hemibagrus punctatus (Jerdon, 1849) and comparative analysis for insights into the phylogeny of hemibagrids through mitogenomic approach.

BACKGROUND: Hemibagrus punctatus (Jerdon, 1849) is a critically endangered bagrid catfish endemic to the Western Ghats of India, whose population is declining due to anthropogenic activities. The current study aims to compare the mitogenome of H. punctatus with that of other Bagrid catfishes and provide insights into their evolutionary relationships. METHODS AND RESULTS: Samples were collected from Hemmige Karnataka, India. In the present study, the mitogenome of H. punctatus was successfully assembled, and its phylogenetic relationships with other Bagridae species were studied. The total genomic DNA of samples was extracted following the phenol-chloroform isoamyl alcohol method. Samples were sequenced, and the Illumina paired-end reads were assembled to a contig length of 16,517 bp. The mitochondrial genome was annotated using MitoFish and MitoAnnotator (Iwasaki et al., 2013). A robust phylogenetic analysis employing NJ (Maximum composite likelihood) and ASAP methods supports the classification of H. punctatus within the Bagridae family, which validates the taxonomic status of this species. In conclusion, this research enriches our understanding of H. punctatus mitogenome, shedding light on its evolutionary dynamics within the Bagridae family and contributing to the broader knowledge of mitochondrial genes in the context of evolutionary biology. CONCLUSIONS: The study's findings contribute to a better understanding of the mitogenome of H. punctatus and provide insights into the evolutionary relationships within other Hemibagrids.

Questioning inbreeding: Could outbreeding affect productivity in the North African catfish in Thailand?

The North African catfish (Clarias gariepinus) is a significant species in aquaculture, which is crucial for ensuring food and nutrition security. Their high adaptability to diverse environments has led to an increase in the number of farms that are available for their production. However, long-term closed breeding adversely affects their reproductive performance, leading to a decrease in production efficiency. This is possibly caused by inbreeding depression. To investigate the root cause of this issue, the genetic diversity of captive North African catfish populations was assessed in this study. Microsatellite genotyping and mitochondrial DNA D-loop sequencing were applied to 136 catfish specimens, collected from three populations captured for breeding in Thailand. Interestingly, extremely low inbreeding coefficients were obtained within each population, and distinct genetic diversity was observed among the three populations, indicating that their genetic origins are markedly different. This suggests that outbreeding depression by genetic admixture among currently captured populations of different origins may account for the low productivity of the North African catfish in Thailand. Genetic improvement of the North African catfish populations is required by introducing new populations whose origins are clearly known. This strategy should be systematically integrated into breeding programs to establish an ideal founder stock for selective breeding.

Molecular and SEM studies on Thaparocleidus vistulensis (Siwak, 1932) (Monopisthocotyla, Ancylodiscoididae).

Presenting new molecular and scanning electron microscope (SEM) features, this study gives additional data to the better knowledge of Thaparocleidus vistulensis (Siwak, 1932) (Monopisthocotyla, Ancylodiscoididae), a parasite of the European catfish Silurus glanis Linnaeus, 1758 (Siluriformes, Siluridae) cultured in a commercial fish farm in Hungary. In addition, notes on the early development of sclerotized anchors are also provided. The main morphological difference of T. vistulensis compared to other congeneric species is associated with the male copulatory organ, which exhibits 5-7 loops in the middle of the penis length and a long open V-shaped sclerotized accessory piece, dividing terminally into two parts, securing the terminal part of the penis tube. The present study provides for the first time molecular characterization data based on the 2694 bp long nucleotide sequence of rDNA (ITS1, 5.8S, ITS2, and flanked with partial 18S and partial 28S) submitted in GenBank with the accession number OR916383. A phylogenetic tree based on ITS1 sequences supports a well-defined clade including T. vistulensis, forming a sister group with T. siluri, a species-specific monopisthocotylan parasite to S. glanis. The morphological characterization of T. vistulensis, especially for the male copulatory organ, together with the molecular data in the present study, extends knowledge about this monopisthocotylan species and provides new information for future phylogeny studies.

Mitogenomic phylogeny, biogeography, and cryptic divergence of the genus Silurus (Siluriformes: Siluridae).

The genus Silurus, an important group of catfish, exhibits heterogeneous distribution in Eurasian freshwater systems. This group includes economically important and endangered species, thereby attracting considerable scientific interest. Despite this interest, the lack of a comprehensive phylogenetic framework impedes our understanding of the mechanisms underlying the extensive diversity found within this genus. Herein, we analyzed 89 newly sequenced and 20 previously published mitochondrial genomes (mitogenomes) from 13 morphological species to reconstruct the phylogenetic relationships, biogeographic history, and species diversity of Silurus. Our phylogenetic reconstructions identified eight clades, supported by both maximum-likelihood and Bayesian inference. Sequence-based species delimitation analyses yielded multiple molecular operational taxonomic units (MOTUs) in several taxa, including the Silurus asotus complex (four MOTUs) and Silurus microdorsalis (two MOTUs), suggesting that species diversity is underestimated in the genus. A reconstructed time-calibrated tree of Silurus species provided an age estimate of the most recent common ancestor of approximately 37.61 million years ago (Ma), with divergences among clades within the genus occurring between 11.56 Ma and 29.44 Ma, and divergences among MOTUs within species occurring between 3.71 Ma and 11.56 Ma. Biogeographic reconstructions suggested that the ancestral area for the genus likely encompassed China and the Korean Peninsula, with multiple inferred dispersal events to Europe and Central and Western Asia between 21.78 Ma and 26.67 Ma and to Japan between 2.51 Ma and 18.42 Ma. Key factors such as the Eocene-Oligocene extinction event, onset and intensification of the monsoon system, and glacial cycles associated with sea-level fluctuations have likely played significant roles in shaping the evolutionary history of the genus Silurus.

Genetic Structure and Diversity of Hatchery and Wild Populations of Yellow Catfish Tachysurus fulvidraco (Siluriformes: Bagridae) from Korea.

Yellow catfish Tachysurus fulvidraco is an important commercial fish species in South Korea. However, due to their current declines in its distribution area and population size, it is being released from hatchery populations into wild populations. Hatchery populations also produced from wild broodstocks are used for its captive breeding. We reported 15 new microsatellite DNA markers of T. fulvidraco to identify the genetic diversity and structure of its hatchery and wild populations, providing baseline data for useful resource development strategies. The observed heterozygosity of the hatchery populations ranged from 0.816 to 0.873, and that of the wild populations ranged from 0.771 to 0.840. Their inbreeding coefficient ranged from -0.078 to 0.024. All populations experienced a bottleneck (p < 0.05), with effective population sizes ranging from 21 to infinity. Their gene structure was divided into two groups with STRUCTURE results of K = 2. It was confirmed that each hatchery population originated from a different wild population. This study provides genetic information necessary for the future development and conservation of fishery resources for T. fulvidraco.

Temporal analysis of genetic diversity and gene flow in the threatened catfish Pseudoplatystoma magdaleniatum from a dammed neotropical river.

The striped catfish Pseudoplatystoma magdaleniatum is a large-sized migratory species from the north Andes region, endemic to Magdalena basin and one of the major fishery resources. Despite the estimated reduction of over 80% of the fisheries production of this species throughout the basin in recent decades, its population in the lower Magdalena-Cauca basin showed healthy genetics after molecular analyses. However, the current conservation status of this species and several habitat disturbances demand the re-evaluation of its population genetics to infer evolutionary risks and assess potential changes. This work analyzed a total of 164 samples from the Cauca River collected downstream the Ituango Dam between 2019-2021 using species-specific microsatellite markers to compare the genetic diversity and structure in samples collected between 2010-2014 from the lower Magdalena-Cauca basin, previously analyzed. Our results showed a relatively stable panmictic population over time (4 to 10 years), with high genetic diversity and evidence of recent bottleneck. Promoting habitat connectivity to conserve gene flow, characterizing diversity and genetic structure over the entire basin, and integrating the results with future monitoring are important aspects for the management planning for P. magdaleniatum in the Magdalena-Cauca basin.

Decoding the molecular phylogenetics of ornamental catfishes (siluriformes) of North East India using DNA barcoding approach.

BACKGROUND: Catfishes (order Siluriformes) are among the most diverse and widely distributed fish groups in the world. They are not only used for human consumption but are also a major part of the ornamental fish trade. Being a Biodiversity Hotspot, the North Eastern Region of India is home to a diverse population of ornamental fishes. Catfishes contain a humongous number of species; in this study, the authors have tried to elucidate the phylogenetic relationship of some important ornamental catfishes found in North East India using DNA barcodes. METHODS AND RESULTS: In this study, we have tried to explore the phylogenetic history of 13 species (41 specimens) of ornamental catfishes spanning 12 genera and 9 families of Siluriformes using DNA barcoding. Pairwise genetic distances using Kimura 2-Parameter (K2P) were calculated at intra-specific and inter-specific levels. A Neighbor-Joining tree was constructed to understand the phylogenetic relationship among the nine different catfish families. All the specimens under this study clustered with their respective species under the same family and formed three sub-clades. However, Olyra longicaudata, belonging to the Bagridae family, did not cluster with other species from the same family. In this study, the authors have suggested a revision of the classification of O. longicaudata back to its original family, Olyridae. CONCLUSIONS: In this study, the maximum intraspecific genetic distance of 0.03 and the minimum interspecific genetic distance of 0.14 were observed among the species. Therefore, it is evident that there is a barcoding gap among the species, which helped in the correct identification of the species. Thus, DNA barcoding helped complement the phenetic approach and also revealed a different phylogenetic relationship among the catfishes belonging to the Bagridae family.

Molecular, behavioral, and growth responses of juvenile yellow catfish (Tachysurus fulvidraco) exposed to carbamazepine.

Carbamazepine (CBZ) is an anticonvulsant medication used to treat epilepsy and bipolar disorder. Due to its persistence and low removal rate in wastewater treatment plants, it is frequently detected in the environment, raising concerns regarding its potential adverse effects on aquatic organisms and ecosystems. In this study, we aimed to assess the impact of CBZ on the behavior and growth of juvenile yellow catfish Tachysurus fulvidraco, a native and economically important species in China. Fish were exposed to CBZ at three concentrations of 1, 10, or 100 µg/L for 14 days. The fish exposed to 10 and 100 µg/L of CBZ exhibited decreased feeding, and a significant increase in cannibalistic tendencies was observed in fish exposed to 100 µg/L CBZ. Acetylcholinesterase activity was increased in the brain of fish exposed to 100 µg/L CBZ. CBZ also inhibited the growth of yellow catfish. To better elucidate mechanisms of toxicity, transcriptomics was conducted in both the brain and liver. In the brain, gene networks associated with neurotransmitter dysfunction were altered by CBZ, as well as networks associated with mitochondrial dysfunction and metabolism. In the liver, gene networks associated with the immune system were altered by CBZ. The current study improves comprehension of the sub-lethal effects of CBZ and reveals novel insight into molecular and biochemical pathways disrupted by CBZ, identifying putative key events associated with reduced growth and altered behavior. This study emphasizes the necessity for improved comprehension of the effects of pharmaceutical contaminants on fish at environmentally relevant levels.

New records of native and introduced fish species in a river basin of Western Ecuador, the Chocó-Darien Ecoregion, using DNA barcoding.

DNA barcoding, based on mitochondrial markers, is widely applied in species identification and biodiversity studies. The aim of this study was to establish a barcoding reference database of fishes inhabiting the Cube River from Western Ecuador in the Chocó-Darien Global Ecoregion (CGE), a threatened ecoregion with high diversity and endemism, and evaluate the applicability of using barcoding for the identification of fish species. Barcode sequences were obtained from seven orders, 17 families, 23 genera and 26 species, which were validated through phylogenetic analysis, morphological measurements, and literature review. Our results showed that 43% of fish species in this region are endemic, confirmed the presence of known species in the area, and included the addition of three new records of native (Hoplias microlepis, Rhamdia guatemalensis and Sicydium salvini) and an introduced species (Xiphophorus maculatus) to Ecuador. In addition, eight species were barcoded for the first time. Species identification based on barcoding and morphology showed discrepancy with species lists from previous studies in the CGE, suggesting that the current baseline of western fishes of Ecuador is still incomplete. Because this study analyzed fishes from a relatively small basin (165 km2), more molecular-based studies focusing on fish are needed to achieve a robust sequence reference library of species inhabiting Western Ecuador. The new sequences of this study will be useful for future comparisons and biodiversity monitoring, supporting the application of barcoding tools for studying fish diversity in genetically unexplored regions and to develop well-informed conservation programs.

Characterization and function of PTEN-induced putative kinase 1 (PINK1) in process of Zinc alleviates hepatic lipid deposition of yellow catfish (Pelteobagrus fulvidraco).

PTEN-induced putative kinase 1 (PINK1) is a key regulator of mitophagy, however, the relevant information remains poorly understood on aquatic animals. Here, a PINK1 gene was cloned, characterized and functionally studied in yellow catfish. PINK1 encoded a protein containing 570 amino acids, 2 functional domains. High fat (15.66%) fed fish showed a downregulation trend of liver PINK1 expression than that of normal fat (10.14%) group, and was reversed by the addition of Zn. In the in vitro study, high fat (HF) can increase lipid deposition and decrease by addition Zn (HFZ) in hepatocytes, whereas above phenomena reversed by overexpression/interference of PINK1, respectively. In addition, the addition of Zn can significantly affect mitochondrial activity, increase mitophagy, and improve the antioxidant activity of hepatocytes. Together, these findings illustrated that yellow catfish PINK1 is conserve, and it participated in mitochondria control of fish. These findings indicate Zn could alleviate high fat-induced hepatic lipid deposition of fish by activating PINK1-mediated mitophagy and provide basis for further exploring new approach for decreasing lipid deposition in fish products during aquaculture.

Molecular characterization of Aeromonas hydrophila detected in Channa marulius and Sperata sarwari sampled from rivers of Punjab in Pakistan.

Aeromonas hydrophila is one of the major pathogenic bacteria responsible for causing severe outbreaks at fish farms and is also a major global public health concern. This bacterium harbors many virulence genes. The current study was designed to evaluate the antidrug and virulence potential of A. hydrophila by amplifying its antimicrobial resistance and virulence genes using PCR and examining their effects on fish tissues and organs. A total of 960 fish samples of Channa marulius and Sperata sarwari were collected from four sites of the rivers of the Punjab, Pakistan. A. hydrophila isolates were subjected to biochemical identification and detection of virulence and antimicrobial resistance (AMR) genes by PCR. We retrieved 181 (6.46%) A. hydrophila isolates from C. marulius and 177 (6.25%) isolates from S. sarwari. Amplification through PCR revealed the incidence of virulence genes in 95.7% of isolates in C. marulius and 94.4% in S. sarwari. Similarly, amplification through PCR also revealed occurrence of AMR genes in 87.1% of isolates in C. marulius and 83.9% in S. sarwari. Histopathological examination revealed congestion (5.2%) and hepatocyte necrosis (4.6%) in liver, lamellar fusion (3.3%) and the presence of bacterial colonies (3.7%) in gills, fin erosion (6%), and the presence of biofilms (3.5%) in tail fins of infected fish. Phylogenetic tree analysis of 16S rRNA and gyrB gene of A. hydrophila revealed 100% and 97% similarity, respectively, with 16S rRNA gene and gyrB of A. hydrophila isolated in previous studies. The results of antimicrobial susceptibility testing showed that all isolates demonstrated resistance to sulfamethoxazole, ampicillin, neomycin, and norfloxacin, while susceptibility to gentamicin, chloramphenicol, and tetracycline, and intermediate resistance was observed against cefotaxime. The results concluded that examined fish samples were markedly contaminated with virulent and multidrug strains of A. hydrophila which may be of a potential health risk. The study emphasizes the responsible antimicrobial use in aquaculture and the urgent need for effective strategies to control the spread of virulence and antimicrobial resistance genes in A. hydrophila.

Occurrence and characterization of Proteus mirabilis from infected farmed African catfish in Ogun State, Nigeria.

BACKGROUND: Bacterial pathogens are the causative agents of some of the most serious disease problems in cultured fish causing mortalities and severe economic losses. This study was conducted to determine the occurrence and characterization of Proteus mirabilis from infected farmed African catfish in Ogun State, Nigeria. METHODOLOGY: The bacteria were isolated from diseased farmed African catfish (Clarias gariepinus, n=128) with clinical signs of skin haemorrhages, ulceration, and ascites purposively sampled from farms within three senatorial districts namely Ogun East (OE; n=76), Ogun Central (OC; n=30) and Ogun West (OW; n=22) in Ogun State. The isolates were identified based on morphological characteristics, biochemical tests, and 16S rRNA gene characterisation. The 16S rRNA gene sequences were analysed using BLAST, submitted to the NCBI database, and an accession number was generated. RESULTS: The occurrence of Proteus mirabilis in infected Clarias gariepinus was 13.16%, 25%, and 31.25% in OE, OC, and OW, respectively. A significantly higher incidence was recorded in OW compared to other areas. All the Proteus mirabilis isolates were motile, gram-negative, short rod, non-lactose fermenter bacteria that showed positive catalase reactions, negative oxidase, and positive for methyl-red. The Proteus mirabilis isolates (OP 594726.1) were closely related to isolates from Pakistan, Italy, and India CONCLUSIONS: We conclude that Proteus mirabilis colonises farmed Clarias gariepinus in Ogun State, Nigeria and the identified strain showed an evolutionary relationship with known pathogenic NCBI reference strains from other countries.

Genetic diversity, spatial connectivity, and population structure of Asian silurid catfish Wallago attu (Bloch and Schneider, 1801) in the Ganga River System: insights from mitochondrial DNA analysis.

BACKGROUND: The Ganga River System (GRS) is a biodiversity hotspot, its ecological richness is shaped by a complex geological history. In this study, we examined the genetic diversity, spatial connectivity, and population structure of the Asian Silurid catfish, Wallago attu, across seven tributaries of the GRS. METHODS AND RESULTS: We employed three mitochondrial DNA (mtDNA) regions: cytochrome c oxidase subunit I (COXI), cytochrome b (Cyt b), and control region (CR). Our comprehensive dataset encompassed 2420 bp of mtDNA, derived from 176 W. attu individuals across 19 sampling sites within the seven rivers of GRS. Our findings revealed high gene diversity (Hd:0.99) within W. attu populations. Analysis of Molecular Variance (AMOVA) highlighted that maximum genetic variations were attributed within the populations, and the observed genetic differentiation among the seven populations of W. attu ranged from low to moderate. Network analysis uncovered the presence of three distinct genetic clades, showing no specific association with seven studied rivers. Bayesian skyline plots provided insights into the demographic history of W. attu, suggesting a recent population expansion estimated to have occurred approximately 0.04 million years ago (mya) during the Pleistocene epoch. CONCLUSIONS: These results significantly enhance our understanding of the genetic diversity and spatial connectivity of W. attu, serving as a vital foundation for developing informed conservation strategies and the sustainable management of this economically valuable resource within the Ganga River System.

A reference genome for the Andean cavefish Trichomycterus rosablanca (Siluriformes, Trichomycteridae): Building genomic resources to study evolution in cave environments.

Animals living in caves are of broad relevance to evolutionary biologists interested in understanding the mechanisms underpinning convergent evolution. In the Eastern Andes of Colombia, populations from at least two distinct clades of Trichomycterus catfishes (Siluriformes) independently colonized cave environments and converged in phenotype by losing their eyes and pigmentation. We are pursuing several research questions using genomics to understand the evolutionary forces and molecular mechanisms responsible for repeated morphological changes in this system. As a foundation for such studies, here we describe a diploid, chromosome-scale, long-read reference genome for Trichomycterus rosablanca, a blind, depigmented species endemic to the karstic system of the department of Santander. The nuclear genome comprises 1 Gb in 27 chromosomes, with a 40.0× HiFi long-read genome coverage having an N50 scaffold of 40.4 Mb and N50 contig of 13.1 Mb, with 96.9% (Eukaryota) and 95.4% (Actinopterygii) universal single-copy orthologs (BUSCO). This assembly provides the first reference genome for the speciose genus Trichomycterus, serving as a key resource for research on the genomics of phenotypic evolution.

FOXO3/Rab7-Mediated Lipophagy and Its Role in Zn-Induced Lipid Metabolism in Yellow Catfish (Pelteobagrus fulvidraco).

Lipophagy is a selective autophagy that regulates lipid metabolism and reduces hepatic lipid deposition. However, the underlying mechanism has not been understood in fish. In this study, we used micronutrient zinc (Zn) as a regulator of autophagy and lipid metabolism and found that Ras-related protein 7 (rab7) was involved in Zn-induced lipophagy in hepatocytes of yellow catfish Pelteobagrus pelteobagrus. We then characterized the rab7 promoter and identified binding sites for a series of transcription factors, including Forkhead box O3 (FOXO3). Site mutation experiments showed that the -1358/-1369 bp FOXO3 binding site was responsible for Zn-induced transcriptional activation of rab7. Further studies showed that inhibition of rab7 significantly inhibited Zn-induced lipid degradation by lipophagy. Moreover, rab7 inhibitor also mitigated the Zn-induced increase of cpt1α and acadm expression. Our results suggested that Zn exerts its lipid-lowering effect partly through rab7-mediated lipophagy and FA ß-oxidation in hepatocytes. Overall, our findings provide novel insights into the FOXO3/rab7 axis in lipophagy regulation and enhance the understanding of lipid metabolism by micronutrient Zn, which may help to reduce excessive lipid accumulation in fish.

Diversity and genetic structure of freshwater shark Wallago attu: an emerging species of commercial interest.

Pakistan has natural freshwater resources acting as a hotspot for diverse fish fauna. However, this aquatic fauna is declining at an alarming rate due to over-exploitation, habitat degradation, water pollution, climate change, and certain anthropogenic activities. The freshwater shark, Wallago attu, is a popular edible catfish inhabiting these freshwater ecosystems. Habitat degradation, overfishing, and human activities are heavily impacting the natural population of this species. So, sound knowledge about its population structure is necessary for its proper management in natural waters. The current study involves utilizing two mtDNA markers (COI, Cytb) to assess the genetic structure and differentiation among W. attu populations of Pakistani Rivers. Genetic variability analysis indicated a high haplotype (0.343 ± 0.046-0.870 ± 0.023) and low nucleotide diversity (0.0024 ± 0.012-0.0038 ± 0.018) among single and combined gene sequences, respectively. Overall, River Indus was populated with more diverse fauna of Wallago attu as compared to River Chenab and River Ravi. Population pairwise, Fst values (0.40-0.61) were found to be significantly different (p < 0.01) among three Riverine populations based upon combined gene sequences. The gene flow for the combined gene (COI + Cytb) dataset among three populations was less than 1.0. The transition/transversion bias value R (0.58) was calculated for testing of neutral evolution, and it declared low genetic polymorphism among natural riverine populations of Wallago attu. The current study's findings would be meaningful in planning the management and conservation of this economically important catfish in future.

Differential expression and alternative splicing analyses of multiple tissues reveal albinism-associated genes in the Wels catfish (Silurus glanis).

Albinism is a widespread departure from a typical body colouration due to altered melanin production. The Wels catfish (Silurus glanis) is among the largest freshwater fish species in the world, and albino individuals occur both in the wild and in aquaculture. Here, we performed transcriptome-wide analysis of albino and normally pigmented S. glanis using four tissues (skin, dorsal fin, whole eye and liver) to identify genes associated with albinism by exploring patterns of differential expression (DE) and differential alternative splicing (DAS). Multi-tissue analyses revealed a large number of genes in skin (n = 1355) and fin (n = 614) tissue associated with the albino phenotype in S. glanis, while the number of DE genes in eye and liver tissues was lower (n = 188, n = 189, respectively). Several DE genes across multiple tissues were detected as the most promising candidates (e.g., hsp4, hsp90b1, raph1, uqcrfs1, adcy-family and wnt-family) potentially causally linked to the albino phenotype in Wels catfish. Moreover, our findings supported earlier observations of physiological differences between albino and normally pigmented individuals, particularly in energy metabolism and immune response. In contrast, there were only a few pigmentation-related genes observed among DAS genes (4 in skin, 2 in fin), the overlap between DAS and DE genes was low (n = 25) and did not include known pigmentation-related genes. This suggests that DAS and DE in Wels catfish are, to a large extent, independent processes, and the observed alternative splicing cases are probably not causally linked with albinism in S. glanis. This work provides the first transcriptome-wide multi-tissue insights into the albinism of Wels catfish and serves as a valuable resource for further understanding the genetic mechanisms of pigmentation in fish.

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.

TBK1 upregulates the interferon response against virus by the TBK1-IRF3/7 axis in yellow catfish (Pelteobagrus fulvidraco).

Yellow catfish (Pelteobagrus fulvidraco) is an important economic species of freshwater fish, widely distributed in China. Recently, viral diseases of yellow catfish have been identified in Chian (Hubei province), arising more attention to the viral immunity in P. fulvidraco. Tumor necrosis factor (TNF) receptor-associated factor NF-κB activator (TANK)-binding kinase 1 (TBK1) plays an essential role in IFN production and innate antiviral immunity. In the present study, we characterized the P. fulvidraco TBK1 (PfTBK1) and reported its function in interferon response. The full-length open reading frame (ORF) is 2184 bp encoding a protein with 727 amino acids, which is composed of four conserved domains, including KD, ULD, CCD1, and CCD2, similar to TBK1 in other species. Pftbk1 was widely expressed in all detected tissues by qPCR and was not inducible by the spring viremia of carp virus (SVCV), a single-strand RNA virus. In addition, the cellular distribution indicated that PfTBK1 was only located in the cytoplasm. Moreover, PfTBK1 induced strong IFN promoter activities through the Jak-stat pathway, and PfTBK1 interacted with and significantly phosphorylated IFN regulatory factor 3/7 (IRF3/7) in P. fulvidraco, promoting the nuclear translocation of pfIRF3 and PfIRF7, and PfTBK1 upregulated IFN response by PfTBK1-PfIRF3/7 axis. Above all, PfTBK1 triggered IFN response and strongly inhibited the replication of SVCV in EPC cells through induction of IFN downstream IFN-stimulated genes (ISGs). Summarily, this work reveals that PfTBK1 plays a positive regulatory role in IFN induction through the TBK1-IRF3/7 axis, laying a foundation for further exploring the molecular mechanism of the antiviral process in P. fulvidraco.