Genome sequencing of Coryphaenoides yaquinae reveals convergent and lineage-specific molecular evolution in deep-sea adaptation.

Abyssal (3501-6500 m) and hadal (>6500 m) fauna evolve under harsh abiotic stresses, characterized by high hydrostatic pressure, darkness and food shortage, providing unique opportunities to investigate mechanisms underlying environmental adaptation. Genomes of several hadal species have recently been reported. However, the genetic adaptation of deep sea species across a broad spectrum of ocean depths has yet to be thoroughly investigated, due to the challenges imposed by collecting the deep sea species. To elucidate the correlation between genetic innovation and vertical distribution, we generated a chromosome-level genome assembly of the macrourids Coryphaenoides yaquinae, which is widely distributed in the abyssal/hadal zone ranging from 3655 to 7259 m in depth. Genomic comparisons among shallow, abyssal and hadal-living species identified idiosyncratic and convergent genetic alterations underlying the extraordinary adaptations of deep-sea species including light perception, circadian regulation, hydrostatic pressure and hunger tolerance. The deep-sea fishes (Coryphaenoides Sp. and Pseudoliparis swirei) venturing into various ocean depths independently have undergone convergent amino acid substitutions in multiple proteins such as rhodopsin 1, pancreatic and duodenal homeobox 1 and melanocortin 4 receptor which are known or verified in zebrafish to be related with vision adaptation and energy expenditure. Convergent evolution events were also identified in heat shock protein 90 beta family member 1 and valosin-containing protein genes known to be related to hydrostatic pressure adaptation specifically in fishes found around the hadal range. The uncovering of the molecular convergence among the deep-sea species shed new light on the common genetic innovations required for deep-sea adaptation by the fishes.

Identification of two miRNAs regulating cardiomyocyte proliferation in an Antarctic icefish.

The hemoglobinless Antarctic icefish develop large hearts to compensate for reduced oxygen-carrying capacity, which serves as a naturally occurred model to explore the factors regulating cardiogenesis. Through miRNAome and microRNAome comparisons between an icefish (Chionodraco hamatus) and two red-blooded notothenioids, we discovered significant upregulation of factors in the BMP signaling pathways and altered expression of many miRNAs, including downregulation of 14 miRNAs in the icefish heart. Through knocking down of these miRNAs, we identified two of them, miR-458-3p and miR-144-5p, involved in enlarged heart development. The two miRNAs were found to regulate cardiomyocyte proliferation by targeting bone morphogenetic protein-2 (bmp2). We further validated that activation of the miRNA-bmp2 signaling in the fish heart could be triggered by hypoxic exposure. Our study suggested that a few miRNAs play important roles in the hypoxia-induced cardiac remodeling of the icefish which shed new light on the mechanisms regulating cardiomyocyte proliferation in heart.

CRISPR/Cas9-induced LEAP2 and GHSR1a knockout mutant zebrafish displayed abnormal growth and impaired lipid metabolism.

Investigating the principles of fish fat deposition and conducting related research are current focal points in fish nutrition. This study explores the endocrine regulation of LEAP2 and GHSR1a in zebrafish by constructing mutantmodels andexamining the effects of the endocrine factors LEAP2 and its receptor GHSR1a on zebrafish growth, feeding, and liver fat deposition. Compared to the wild type (WT), the mutation of LEAP2 results in increased feeding and decreased swimming in zebrafish. The impact is more pronounced in adult female zebrafish, characterized by increased weight, length, width, and accumulation of lipid droplets in the liver.Incontrast, deficiency in GHSR1a significantly reduces the growth of male zebrafish and markedly decreases liver fat deposition.These research findings indicate the crucial roles of LEAP2 and GHSR1a in zebrafish feeding, growth, and intracellular fat metabolism. This study, for the first time, investigated the endocrine metabolic regulation functions of LEAP2 and GHSR1a in the model organism zebrafish, providing initial insights into their effects and potential mechanisms on zebrafish fat metabolism.

Metabolomic-based analysis reveals bile acid-mediated ovarian failure induced by low temperature in zebrafish.

As ectotherms, fish are highly sensitive to temperature fluctuations, which can profoundly impact their reproductive cycles. In this study, we investigated the fertility and histological characteristics of zebrafish ( Danio rerio) ovaries exposed to a temperature gradient ranging from the thermopreferendum temperature of the species, 27°C, to lower temperatures of 22°C, 20°C, and 13°C over a period of two weeks. Comparative metabolomic (six biological replicates for each temperature) and transcriptomic (four biological replicates for each temperature) analyses were conducted under the four temperature conditions. Results indicated that lower temperatures inhibited oocyte development and differential metabolites were involved in steroid hormone production, antioxidant function, and lipid and protein catabolism. Disrupted reproductive hormones, increased proteolysis, and lipid degradation significantly impeded oocyte development and egg maturation. Notably, a significant increase in bile acid content was noted in the ovaries of the cold-treated fish, indicating that bile acids play a critical role in ovarian failure. Overall, these findings provide valuable insights into the mechanisms governing the reproductive response of fish to cold stress.

Deletion of Asb15b gene can lead to a significant decrease in zebrafish intermuscular bone.

Intermuscular bones, which are present in numerous economically significant fish species, have a negative impact on the development of aquaculture. The Asb15b gene, primarily expressed in skeletal muscle, plays a crucial role in regulating protein turnover and the development of muscle fibers. It stimulates protein synthesis and controls the differentiation of muscle fibers. In this study, we employed CRISPR/Cas9 technology to generate homozygous zebrafish strains with 7 bp and 49 bp deletions in the Asb15b gene. Subsequent analyses using skeleton staining demonstrated a substantial reduction in the number of intermuscular bones in adult Asb15b-/- -7 bp and Asb15b-/- -49 bp mutants compared to the wild-type zebrafish, with decreases of 30 % (P < 0.001) and 40 % (P < 0.0001), respectively. Histological experiments further revealed that the diameter and number of muscle fibers in adult Asb15b-/- mutants did not exhibit significant changes when compared to wild-type zebrafish. Moreover, qRT-PCR experiments demonstrated significant differences in the expression of bmp6 and runx2b genes, which are key regulators of intermuscular bone development, during different stages of intermuscular bone development in Asb15b-/- mutants. This study strongly suggests that the Asb15b gene plays a crucial role in regulating intermuscular bone development in fish and lays the groundwork for further exploration of the role of the Asb15b gene in zebrafish intermuscular bone development.

Differential tissue immune stimulation through immersion in bacterial and viral agonists in the Antarctic Notothenia rossii.

The genome evolution of Antarctic notothenioids has been modulated by their extreme environment over millennia and more recently by human-caused constraints such as overfishing and climate change. Here we investigated the characteristics of the immune system in Notothenia rossii and how it responds to 8 h immersion in viral (Poly I:C, polyinosinic: polycytidylic acid) and bacterial (LPS, lipopolysaccharide) proxies. Blood plasma antiprotease activity and haematocrit were reduced in Poly I:C-treated fish only, while plasma protein, lysozyme activity and cortisol were unchanged with both treatments. The skin and duodenum transcriptomes responded strongly to the treatments, unlike the liver and spleen which had a mild response. Furthermore, the skin transcriptome responded most to the bacterial proxy (cell adhesion, metabolism and immune response processes) and the duodenum (metabolism, response to stress, regulation of intracellular signal transduction, and immune system responses) to the viral proxy. The differential tissue response to the two proxy challenges is indicative of immune specialisation of the duodenum and the skin towards pathogens. NOD-like and C-type lectin receptors may be central in recognising LPS and Poly I:C. Other antimicrobial compounds such as iron and selenium-related genes are essential defence mechanisms to protect the host from sepsis. In conclusion, our study revealed a specific response of two immune barrier tissue, the skin and duodenum, in Notothenia rossii when exposed to pathogen proxies by immersion, and this may represent an adaptation to pathogen infective strategies.

The First High-Quality Genome Assembly of Freshwater Pearl Mussel Sinohyriopsis cumingii: New Insights into Pearl Biomineralization.

China leads the world in freshwater pearl production, an industry in which the triangle sail mussel (Sinohyriopsis cumingii) plays a pivotal role. In this paper, we report a high-quality chromosome-level genome assembly of S. cumingii with a size of 2.90 Gb-the largest yet reported among bivalves-and 89.92% anchorage onto 19 linkage groups. The assembled genome has 37,696 protein-coding genes and 50.86% repeat elements. A comparative genomic analysis revealed expansions of 752 gene families, mostly associated with biomineralization, and 237 genes under strong positive selection. Notably, the fibrillin gene family exhibited gene family expansion and positive selection simultaneously, and it also exhibited multiple high expressions after mantle implantation by transcriptome analysis. Furthermore, RNA silencing and an in vitro calcium carbonate crystallization assay highlighted the pivotal role played by one fibrillin gene in calcium carbonate deposition and aragonite transformation. This study provides a valuable genomic resource and offers new insights into the mechanism of pearl biomineralization.

Molecular characterization, expression analysis and subcellular location of the members of STAT family from spotted seabass (Lateolabrax maculatus).

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway is a pervasive intracellular signal transduction pathway, involving in biological processes such as cell proliferation, differentiation, apoptosis and immune regulation. In this study, seven STAT genes, STAT1, STAT1-like, STAT2, STAT3, STAT4, STAT5a and STAT5b, were identified and characterized in spotted seabass (Lateolabrax maculatus). Analyses of multiple sequence alignment, genomic organization, phylogeny and conserved synteny were conducted to infer the evolutionary conservation of these genes in the STAT family. The results of the bioinformatics analysis assumed that STAT1 and STAT1-like might be homologous to STAT1a and STAT1b, respectively. Furthermore, the expression of the seven genes were detected in eight tissues of healthy spotted seabass, which revealed that they were expressed in a variety of tissues, mainly in gill, spleen and muscle, and extremely under-expression in liver. The expression of the seven genes in gill, head-kidney, spleen and intestine were significantly induced by lipopolysaccharide (LPS) or Edwardsiella tarda challenge. The expression of most of the LmSTATs were up-regulated, and the highest expression levels at 12 h after LPS stimulation, however, the LmSTATs were down-regulated by E. tarda infection. The results of subcellular localization show that the native LmSTAT1, LmSTAT1-like, LmSTAT2, LmSTAT3 and LmSTAT5a were localized in the cytoplasm, but they were translocated into the nucleus after LPS stimulation. Whereas, LmSTAT4 and LmSTAT5b were translocation into the nucleus whether with LPS stimulation or not. Overall, this is the first study to systematically revealed the localization of STAT members in fish, and indicated that LmSTATs participate in the process of protecting the host from pathogens invasion in the form of entry into nucleus.

New insights into the antimicrobial mechanism of LEAP2 mutant zebrafish under Aeromonas hydrophila infection using transcriptome analysis.

Liver-expressed antimicrobial peptide 2 (LEAP2) is a blood-derived antimicrobial peptide expressed predominantly in the liver. Although LEAP2 has been reported to exert antimicrobial effects in various fish species, its antimicrobial mechanism is not entirely understood. Zebrafish is an intensively developing animal model for studying bacterial diseases. In this study, we used zebrafish to identify the role of LEAP2 in bacterial infection. We found that knockout of LEAP2 in zebrafish led to a higher bacterial burden and mortality. To further investigate the effect of LEAP2 mutation on the immune system, we conducted a comparative transcriptome analysis of zebrafish with a mutant of LEAP2. Based on gene ontologies (GO) enrichment, LEAP2 mutant zebrafish revealed that, compared to wild-type zebrafish, robust responses to bacteria, inflammatory factors, and disrupt immune homeostasis and induct hyperinflammation. Furthermore, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, six immune pathways were identified: Phagosome, NOD-like receptor, ferroptosis, Cytokine-cytokine receptor, Toll-like receptor, and FOXO signalling pathways. Interestingly, besides the liver, muscle, intestine, and eggs are also significantly enriched to the ferroptosis pathway, as revealed using quantitative polymerase chain reaction (qPCR), further confirmed that the effect of LEAP2 mutations on inflammatory factors and ferroptosis-related genes. Most importantly, this is the first report of the zebrafish LEAP2 mutant transcriptome obtained using high-throughput sequencing. Our study employed comparative transcriptome analysis to reveal the inflammatory response and ferroptosis-signalling pathway as a novel potential mechanism of LEAP2 antibacterial activity, laying the foundation for future studies of LEAP2 immune functions.

Tissue-specific temperature dependence of RNA editing levels in zebrafish.

BACKGROUND: RNA editing by adenosine deaminase acting on RNA (ADAR) occurs in all metazoans and fulfils several functions. Here, we examined effects of acclimation temperature (27 °C, 18 °C,13 °C) on editing patterns in six tissues of zebrafish (Danio rerio). RESULTS: Sites and total amounts of editing differed among tissues. Brain showed the highest levels, followed by gill and skin. In these highly edited tissues, decreases in temperatures led to large increases in total amounts of editing and changes in specific edited sites. Gene ontology analysis showed both similarities (e.g., endoplasmic reticulum stress response) and differences in editing among tissues. The majority of edited sites were in transcripts of transposable elements and the 3'UTR regions of protein coding genes. By experimental validation, translation efficiency was directly related to extent of editing of the 3'UTR region of an mRNA. CONCLUSIONS: RNA editing increases 3'UTR polymorphism and affects efficiency of translation. Such editing may lead to temperature-adaptive changes in the proteome through altering relative amounts of synthesis of different proteins.

Identification of antibacterial activity of LEAP2 from Antarctic icefish Chionodraco hamatus.

Liver-expressed antimicrobial peptide 2 (LEAP2) is a small peptide, which is consisted of signal peptide, pro-peptide and the bioactive mature peptide. Mature LEAP2 is an antibacterial peptide with four highly conserved cysteines forming two intramolecular disulfide bonds. Chionodraco hamatus, an Antarctic notothenioid fish that lives in the coldest water, has white blood unlike most fish of the world. In this study, the LEAP2 coding sequence was cloned from C. hamatus, including a 29 amino acids signal peptide and mature peptide of 46 amino acids. High levels of LEAP2 mRNA were detected in the skin and liver. Mature peptide was obtained by chemical synthesis in vitro, displayed selective antimicrobial activities against Escherichia coli, Aeromonas hydrophila, Staphylococcus aureus and Streptococcus agalactiae. Liver-expressed antimicrobial peptide 2 showed bactericidal activity by destroying the cell membrane integrity and robustly combined with bacterial genomic DNA. In addition, overexpression of the Tol-LEAP2-EGFP in zebrafish larva showed stronger antimicrobial activity in C. hamatus than in zebrafish, accompanied by lower bacterial load and expression of pro-inflammatory factors. This is the first demonstration of the antimicrobial activity of LEAP2 from C. hamatus, which is of useful value in improving resistance to pathogens.

Assessment of Nonalcoholic Fatty Liver Disease Symptoms and Gut-Liver Axis Status in Zebrafish after Exposure to Polystyrene Microplastics and Oxytetracycline, Alone and in Combination.

BACKGROUND: Environmental pollution may give rise to the incidence and progression of nonalcoholic fatty liver disease (NAFLD), the most common cause for chronic severe liver lesions. Although knowledge of NAFLD pathogenesis is particularly important for the development of effective prevention, the relationship between NAFLD occurrence and exposure to emerging pollutants, such as microplastics (MPs) and antibiotic residues, awaits assessment. OBJECTIVES: This study aimed to evaluate the toxicity of MPs and antibiotic residues related to NAFLD occurrence using the zebrafish model species. METHODS: Taking common polystyrene MPs and oxytetracycline (OTC) as representatives, typical NAFLD symptoms, including lipid accumulation, liver inflammation, and hepatic oxidative stress, were screened after 28-d exposure to environmentally realistic concentrations of MPs (0.69mg/L) and antibiotic residue (3.00µg/L). The impacts of MPs and OTC on gut health, the gut-liver axis, and hepatic lipid metabolism were also investigated to reveal potential affecting mechanisms underpinning the NAFLD symptoms observed. RESULTS: Compared with the control fish, zebrafish exposed to MPs and OTC exhibited significantly higher levels of lipid accumulation, triglycerides, and cholesterol contents, as well as inflammation, in conjunction with oxidative stress in their livers. In addition, a markedly smaller proportion of Proteobacteria and higher ratios of Firmicutes/Bacteroidetes were detected by microbiome analysis of gut contents in treated samples. After the exposures, the zebrafish also experienced intestinal oxidative injury and yielded significantly fewer numbers of goblet cells. Markedly higher levels of the intestinal bacteria-sourced endotoxin lipopolysaccharide (LPS) were also detected in serum. Animals treated with MPs and OTC exhibited higher expression levels of LPS binding receptor (LBP) and downstream inflammation-related genes while also exhibiting lower activity and gene expression of lipase. Furthermore, MP-OTC coexposure generally exerted more severe effects compared with single MP or OTC exposure. DISCUSSION: Our results suggested that exposure to MPs and OTC may disrupt the gut-liver axis and be associated with NAFLD occurrence. https://doi.org/10.1289/EHP11600.

Toxicities of polystyrene microplastics (MPs) and hexabromocyclododecane (HBCD), alone or in combination, to the hepatopancreas of the whiteleg shrimp, Litopenaeus vannamei.

The hepatopancreas is one of the largest organs playing crucial roles in metabolism and detoxification in crustacean invertebrates. Although toxicities have been increasingly documented for the two ubiquitous pollutants, hexabromocyclododecane (HBCD) and microplastics (MPs), in model animals, little is known about their impacts on the hepatopancreas of crustaceans. To fill this knowledge gap, the effects of MPs and HBCD, alone or in combination, on the hepatopancreas were evaluated in a commercially important crustacean species (the whiteleg shrimp) by histological observation as well as quantification of hepatic lesion-, metabolism-, and detoxification-related parameters. In addition, to reveal potential mechanisms underlying the hepatoxicity observed, the accumulation of HBCD in the shrimp and the status of oxidative stress were also investigated. Our results demonstrated that exposure of the whiteleg shrimp to MPs and HBCD for 4 weeks resulted in evident histological injury in the hepatopancreas and marked elevation in hepatic lesion markers (alanine aminotransferase and aspartate aminotransferase) in the hemolymph. Moreover, both metabolism (activity of phosphofructokinase, contents of lactic acid and adenosine triphosphate, and expression of metabolism-related genes) and detoxification (contents of cytochrome P450, UDP-glucuronosyltransferase, and glutathione, activity of glutathione S-transferase, and expression of detoxification-related genes) were found to be disrupted by the pollutants tested. In addition, exposure to MPs and HBCD also led to alterations in the contents and/or activities of antioxidant enzymes and resulted in oxidative damage to the hepatopancreas (indicated by marked elevation in malondialdehyde content). Furthermore, a significant amount of HBCD accumulated in shrimp treated with HBCD-containing seawater. The data also illustrated that HBCD-MP coexposure was more toxic than single exposure to these pollutants. These findings suggest that MPs and HBCD may exert hepatotoxic impacts on whiteleg shrimp by accumulating in vivo and inducing oxidative stress, which could pose a severe threat to the health of this important crustacean species.

Sex-specific meiosis responses to Gsdf in medaka (Oryzias latipes).

The meiotic entry of undifferentiated germ cells is sexually specific and strictly regulated by the testicular or ovarian environment. Germline stem cells with a set of abnormal sex chromosomes and associated autosomes undergo defective meiotic processes and are eventually eliminated by yet to be defined post-transcriptional modifications. Herein, we report the role of gsdf, a member of BMP/TGFß family uniquely found in teleost, in the regulation of meiotic entry in medaka (Oryzias latipes) via analyses of gametogenesis in gsdf-deficient XX and XY gonads in comparison with their wild-type siblings. Several differentially expressed genes, including the FKB506-binding protein 7 (fkbp7), were significantly upregulated in pubertal gsdf-deficient gonads. The increase in alternative pre-mRNA isoforms of meiotic synaptonemal complex gene sycp3 was visualized using Integrative Genomics Viewer and confirmed by real-time qPCR. Nevertheless, immunofluorescence analysis showed that Sycp3 protein products reduced significantly in gsdf-deficient XY oocytes. Transmission electron microscope observations showed that normal synchronous cysts were replaced by asynchronous cysts in gsdf-deficient testis. Breeding experiments showed that the sex ratio deviation of gsdf-/- XY gametes in a non-Mendelian manner might be due to the non-segregation of XY chromosomes. Taken together, our results suggest that gsdf plays a role in the proper execution of cytoplasmic and nuclear events through receptor Smad phosphorylation and Sycp3 dephosphorylation to coordinate medaka gametogenesis, including sex-specific mitotic divisions and meiotic recombination.

Dusp1 regulates thermal tolerance limits in zebrafish by maintaining mitochondrial integrity.

Temperature tolerance restricts the distribution of a species. However, the molecular and cellular mechanisms that set the thermal tolerance limits of an organism are poorly understood. Here, we report on the function of dual-specificity phosphatase 1 (DUSP1) in thermal tolerance regulation. Notably, we found that dusp1 -/- zebrafish grew normally but survived within a narrowed temperature range. The higher susceptibility of these mutant fish to both cold and heat challenges was attributed to accelerated cell death caused by aggravated mitochondrial dysfunction and over-production of reactive oxygen species in the gills. The DUSP1-MAPK-DRP1 axis was identified as a key pathway regulating these processes in both fish and human cells. These observations suggest that DUSP1 may play a role in maintaining mitochondrial integrity and redox homeostasis. We therefore propose that maintenance of cellular redox homeostasis may be a key mechanism for coping with cellular thermal stress and that the interplay between signaling pathways regulating redox homeostasis in the most thermosensitive tissue (i.e., gills) may play an important role in setting the thermal tolerance limit of zebrafish.

Population genomics of an icefish reveals mechanisms of glacier-driven adaptive radiation in Antarctic notothenioids.

BACKGROUND: Antarctica harbors the bulk of the species diversity of the dominant teleost fish suborder-Notothenioidei. However, the forces that shape their evolution are still under debate. RESULTS: We sequenced the genome of an icefish, Chionodraco hamatus, and used population genomics and demographic modelling of sequenced genomes of 52 C. hamatus individuals collected mainly from two East Antarctic regions to investigate the factors driving speciation. Results revealed four icefish populations with clear reproduction separation were established 15 to 50 kya (kilo years ago) during the last glacial maxima (LGM). Selection sweeps in genes involving immune responses, cardiovascular development, and photoperception occurred differentially among the populations and were correlated with population-specific microbial communities and acquisition of distinct morphological features in the icefish taxa. Population and species-specific antifreeze glycoprotein gene expansion and glacial cycle-paced duplication/degeneration of the zona pellucida protein gene families indicated fluctuating thermal environments and periodic influence of glacial cycles on notothenioid divergence. CONCLUSIONS: We revealed a series of genomic evidence indicating differential adaptation of C. hamatus populations and notothenioid species divergence in the extreme and unique marine environment. We conclude that geographic separation and adaptation to heterogeneous pathogen, oxygen, and light conditions of local habitats, periodically shaped by the glacial cycles, were the key drivers propelling species diversity in Antarctica.

Iron supplementation inhibits hypoxia-induced mitochondrial damage and protects zebrafish liver cells from death.

Acute hypoxia in water has always been a thorny problem in aquaculture. Oxygen and iron play important roles and are interdependent in fish. Iron is essential for oxygen transport and its concentration tightly controlled to maintain the cellular redox homeostasis. However, it is still unclear the role and mechanism of iron in hypoxic stress of fish. In this study, we investigated the role of iron in hypoxic responses of two zebrafish-derived cell lines. We found hypoxia exposed zebrafish liver cells (ZFL) demonstrated reduced expression of Ferritin and the gene fth31 for mitochondrial iron storage, corresponding to reduction of both intracellular and mitochondrial free iron and significant decrease of ROS levels in multiple cellular components, including mitochondrial ROS and lipid peroxidation level. In parallel, the mitochondrial integrity was severely damaged. Addition of exogenous iron restored the iron and ROS levels in cellular and mitochondria, reduced mitochondrial damage through enhancing mitophagy leading to higher cell viability, while treated the cells with iron chelator (DFO) or ferroptosis inhibitor (Fer-1) showed no improvements of the cellular conditions. In contrast, in hypoxia insensitive zebrafish embryonic fibroblasts cells (ZF4), the expression of genes related to iron metabolism showed opposite trends of change and higher mitochondrial ROS level compared with the ZFL cells. These results suggest that iron homeostasis is important for zebrafish cells to maintain mitochondrial integrity in hypoxic stress, which is cell type dependent. Our study enriched the hypoxia regulation mechanism of fish, which helped to reduce the hypoxia loss in fish farming.

MiR-202-3p determines embryo viability during mid-blastula transition.

Developmental growth is an intricate process involving the coordinated regulation of the expression of various genes, and microRNAs (miRNAs) play crucial roles in diverse processes throughout animal development. The mid-blastula transition (MBT) is a developmental milestone when maternal RNAs are cleared and the zygotic genome programmed asynchronous cell division begins to drive embryogenesis. While mechanisms underlying MBT have been intensively revealed, factors regulating cell proliferation at the transition remain largely unknown. We report here a microRNA, miR-202-3p to be a key factor that determines embryonic fate during MBT in zebrafish. A miR-202-3p antagomir specifically terminated embryo development at the mid-blastula stage. In vivo deletion of the miR-202 locus recapitulated the fatal phenotypes, which were rescued only by miR-202-3p or its precursor. Transcriptome comparison revealed >250 RNAs including both maternal and zygotic origins were dysregulated at MBT in the miR-202-/- embryos, corresponding with arrays of homeostatic disorders leading to massive apoptosis. A trio of genes: nfkbiaa, perp and mgll, known to be intimately involved with cell proliferation and survival, were identified as direct targets of miR-202-3p. Importantly, over- or under-expression of any of the trio led to developmental delay or termination at the blastula or gastrula stages. Furthermore, nfkbiaa and perp were shown to inter-regulate each other. Thus, miR-202-3p mediates a regulatory network whose components interact closely during MBT to determine embryonic viability and development.

Transcriptomic and Phenotypic Analysis of CRISPR/Cas9-Mediated gluk2 Knockout in Zebrafish.

As a subtype of kainite receptors (KARs), GluK2 plays a role in the perception of cold in the periphery sensory neuron. However, the molecular mechanism for gluk2 on the cold stress in fish has not been reported. In this article, real-time PCR assays showed that gluk2 was highly expressed in the brain and eyes of adult zebrafish. To study the functions of gluk2, gene knockout was carried out using the CRISPR/Cas9 system. According to RNA-seq analysis, we selected the differentially expressed genes (DEGs) that had significant differences in at least three tissues of the liver, gill, intestine, skin, brain, and eyes. Gene Ontology (GO) enrichment analysis revealed that cry1ba, cry2, per1b, per2, hsp70.1, hsp70.2, hsp70l, hsp90aa1.1, hsp90aa1.2, hspb1, trpv1, slc27a1b, park2, ucp3, and METRNL were significantly enriched in the 'Response to temperature stimulus' pathway. Through behavioral phenotyping assay, the gluk2-/- larval mutant displayed obvious deficiency in cold stress. Furthermore, TUNEL (TdT-mediated dUTP Nick-End Labeling) staining proved that the gill apoptosis of gluk2-/- mutant was increased approximately 60 times compared with the wild-type after gradient cooling to 8 °C for 15 h. Overall, our data suggested that gluk2 was necessary for cold tolerance in zebrafish.

Identification of Antibacterial Activity of Hepcidin From Antarctic Notothenioid Fish.

Hepcidin is a small peptide composed of signal peptide, propeptide, and the bioactive mature peptide from N terminal to C terminal. Mature hepcidin is an antibacterial peptide and iron regulator with eight highly conserved cysteines forming four intramolecular disulfide bonds, giving it a ß sheet hairpin-like structure. Hepcidin homologs are found in a variety of vertebrates, especially fish, and their diversity may be associated with different habitats and different levels of pathogens. Dissostichus mawsoni, an Antarctic notothenioid fish that lives in the coldest water unlike most places of the world, with at least two hepcidin variants with eight cysteines. We confirmed the formation process of activated mature hepcidins from D. mawsoni in Chinese hamster ovary (CHO) cell line, obtained recombinant hepcidin protein from prokaryotes, and characterized its binding ability and antibacterial activity against varying bacteria. The expression of hepcidin in CHO cell line showed that the prepropeptide of Dmhep_8cysV1 and Dmhep_8cysV2 cleavage into smaller mature peptide. The antibacterial assay and flow cytometry showed that Dmhep_8cysV1, Dmhep_8cysV2, and Drhep bound to different bacteria and killed them with different minimum inhibitory concentration. These data suggest that hepcidin plays an important role in the innate immunity of D. mawsoni and is of great value in improving resistance to pathogens.