Successional Changes of Microbial Communities and Host-Microbiota Interactions Contribute to Dietary Adaptation in Allodiploid Hybrid Fish.

Host-microbiota interactions play critical roles in host development, immunity, metabolism, and behavior. However, information regarding host-microbiota interactions is limited in fishes due to their complex living environment. In the present study, an allodiploid hybrid fish derived from herbivorous Megalobrama amblycephala (♀) × carnivorous Culter alburnus (♂) was used to investigate the successional changes of the microbial communities and host-microbiota interactions during herbivorous and carnivorous dietary adaptations. The growth level was not significantly different in any developmental stage between the two diet groups of fish. The diversity and composition of the dominant microbial communities showed similar successional patterns in the early developmental stages, but significantly changed during the two dietary adaptations. A large number of bacterial communities coexisted in all developmental stages, whereas the abundance of some genera associated with metabolism, including Acinetobacter, Gemmobacter, Microbacterium, Vibrio, and Aeromonas, was higher in either diet groups of fish. Moreover, the abundance of phylum Firmicutes, Actinobacteria, and Chloroflexi was positively correlated with the host growth level. In addition, Spearman's correlation analysis revealed that the differentially expressed homologous genes in the intestine associated with cell growth, immunity, and metabolism were related to the dominant gut microbiota. Our results present evidence that host genetics-gut microbiota interactions contribute to dietary adaptation in hybrid fish, which also provides basic data for understanding the diversity of dietary adaptations and evolution in fish.

The subgenomes show asymmetric expression of alleles in hybrid lineages of Megalobrama amblycephala × Culter alburnus.

Hybridization drives rapid speciation by shaping novel genotypic and phenotypic profiles. Genomic incompatibility and transcriptome shock have been observed in hybrids, although this is rarer in animals than in plants. Using the newly sequenced genomes of the blunt snout bream (Megalobrama amblycephala [BSB]) and the topmouth culter (Culter alburnus [TC]), we focused on the sequence variation and gene expression changes in the reciprocal intergeneric hybrid lineages (F1-F3) of BSB × TC. A genome-wide transcriptional analysis identified 145-974 expressed recombinant genes in the successive generations of hybrid fish, suggesting the rapid emergence of allelic variation following hybridization. Some gradual changes of gene expression with additive and dominance effects and various cis and trans regulations were observed from F1 to F3 in the two hybrid lineages. These asymmetric patterns of gene expression represent the alternative strategies for counteracting deleterious effects of the subgenomes and improving adaptability of novel hybrids. Furthermore, we identified positive selection and additive expression patterns in transforming growth factor, beta 1b (tgfb1b), which may account for the morphological variations of the pharyngeal jaw in the two hybrid lineages. Our current findings provide insights into the evolution of vertebrate genomes immediately following hybridization.

Asymmetric expression of homoeologous genes contributes to dietary adaption of an allodiploid hybrid fish derived from Megalobrama amblycephala (♀) × Culter alburnus (♂).

BACKGROUND: Hybridization, which can quickly merge two or more divergent genomes and form new allopolyploids, is an important technique in fish genetic breeding. However, the merged subgenomes must adjust and coexist with one another in a single nucleus, which may cause subgenome interaction and dominance at the gene expression level and has been observed in some allopolyploid plants. In our previous studies, newly formed allodiploid hybrid fish derived from herbivorous Megalobrama amblycephala (♀) × carnivorous Culter alburnus (♂) had herbivorous characteristic. It is thus interesting to further characterize whether the subgenome interaction and dominance derive dietary adaptation of this hybrid fish. RESULTS: Differential expression, homoeolog expression silencing and bias were investigated in the hybrid fish after 70 days of adaptation to carnivorous and herbivorous diets. A total of 2.65 × 108 clean reads (74.06 Gb) from the liver and intestinal transcriptomes were mapped to the two parent genomes based on specific SNPs. A total of 2538 and 4385 differentially expressed homoeologous genes (DEHs) were identified in the liver and intestinal tissues between the two groups of fish, respectively, and these DEHs were highly enriched in fat digestion and carbon metabolism, amino acid metabolism and steroid biosynthesis. Furthermore, subgenome dominance were observed in tissues, with paternal subgenome was more dominant than maternal subgenome. Moreover, subgenome expression dominance controlled functional pathways in metabolism, disease, cellular processes, environment and genetic information processing during the two dietary adaptation processes. In addition, few but sturdy villi in the intestine, significant fat accumulation and a higher concentration of malondialdehyde in the liver were observed in fish fed carnivorous diet compared with fish fed herbivorous diet. CONCLUSIONS: Our results indicated that diet drives phenotypic and genetic variation, and the asymmetric expression of homoeologous genes (including differential expression, expression silencing and bias) may play key roles in dietary adaptation of hybrid fish. Subgenome expression dominance may contribute to uncovering the mechanistic basis of heterosis and also provide perspectives for fish genetic breeding and application.

Maternal effects shape the alternative splicing of parental alleles in reciprocal cross hybrids of Megalobrama amblycephala × Culter alburnus.

BACKGROUND: Maternal effects contribute to adaptive significance for shaping various phenotypes of many traits. Potential implications of maternal effects are the cause of expression diversity, but these effects on mRNA expression and alternative splicing (AS) have not been fully elucidated in hybrid animals. RESULTS: Two reciprocal cross hybrids following hybridization of Megalobrama amblycephala (blunt snout bream, BSB) and Culter alburnus (topmouth culter, TC) were used as a model to investigate maternal effects. By comparing the expression of BSB- and TC- homoeologous genes between the two reciprocal cross hybrids, we identified 49-348 differentially expressed BSB-homoeologous genes and 54-354 differentially expressed TC-homoeologous genes. 2402, 2959, and 3418 AS events between the two reciprocal cross hybrids were detected in Illumina data of muscle, liver, and gonad, respectively. Moreover, 21,577 (TC-homoeologs) and 30,007 (BSB-homoeologs) AS events were found in the 20,131 homoeologous gene pairs of TBF3 based on PacBio data, while 30,561 (TC-homoeologs) and 30,305 (BSB-homoeologs) AS events were found in BTF3. These results further improve AS prediction at the homoeolog level. The various AS patterns in bmpr2a belonging to the bone morphogenetic protein family were selected as AS models to investigate the expression diversity and its potential effects to body shape traits. CONCLUSIONS: The distribution of differentially expressed genes and AS in BSB- and TC-subgenomes exhibited various changes between the two reciprocal cross hybrids, suggesting that maternal effects were the cause of expression diversity. These findings provide a novel insight into mRNA expression changes and AS under maternal effects in lower vertebrates.

Rapid genomic DNA variation in newly hybridized carp lineages derived from Cyprinus carpio (♀) × Megalobrama amblycephala (♂).

BACKGROUND: Distant hybridization can generate changes in phenotypes and genotypes that lead to the formation of new hybrid lineages with genetic variation. In this study, the establishment of two bisexual fertile carp lineages, including the improved diploid common carp (IDC) lineage and the improved diploid scattered mirror carp (IDMC) lineage, from the interspecific hybridization of common carp (Cyprinus carpio, 2n = 100) (♀) × blunt snout bream (Megalobrama amblycephala, 2n = 48) (♂), provided a good platform to investigate the genetic relationship between the parents and their hybrid progenies. RESULT: In this study, we investigated the genetic variation of 12 Hox genes in the two types of improved carp lineages derived from common carp (♀) × blunt snout bream (♂). Hox gene clusters were abundant in the first generation of IDC, but most were not stably inherited in the second generation. In contrast, we did not find obvious mutations in Hox genes in the first generation of IDMC, and almost all the Hox gene clusters were stably inherited from the first generation to the second generation of IDMC. Interestingly, we found obvious recombinant clusters of Hox genes in both improved carp lineages, and partially recombinant clusters of Hox genes were stably inherited from the first generation to the second generation in both types of improved carp lineages. On the other hand, some Hox genes were gradually becoming pseudogenes, and some genes were completely pseudogenised in IDC or IDMC. CONCLUSIONS: Our results provided important evidence that distant hybridization produces rapid genomic DNA changes that may or may not be stably inherited, providing novel insights into the function of hybridization in the establishment of improved lineages used as new fish resources for aquaculture.

Comparative analysis of testis transcriptomes associated with male infertility in triploid cyprinid fish.

Spermatogenesis involves a series of cellular transformations and thousands of regulated genes. Previously, we showed that the triploid fish (3nBY) cannot produce mature spermatozoa. In the present study, evaluation of the testis microstructure revealed that germ cells of 3nBY could develop into round spermatids, but then degenerated, resulting in male infertility. In this study we comparatively analysed the testis transcriptomes from 3nBY and its diploid parent YB and identified a series of differentially expressed genes (DEGs) that were enriched in the Wnt signalling pathway and the apoptotic and ubiquitin-mediated proteolysis processes in 3nBY. Gene ontology functional analyses revealed that some DEGs in 3nBY were directly associated with the process of gamete generation, development and sperm flagellum assembly. In addition, the expression of a number of genes related to meiosis (Inhibitor Of DNA Binding 2 (ID2), Ovo Like Transcriptional Repressor 1 (OVOL1)), mitochondria (ATP1b (ATPase Na+/K+ Transporting Subunit Beta 1), ATP2a (ATPase, Ca++ Transporting, Cardiac Muscle, Slow Twitch 2), ATP5a (ATP Synthase F1 Subunit Alpha), Mitochondrially Encoded Cytochrome C Oxidase I (COX1), NADH Dehydrogenase Subunit 4 (ND4)) and chromatin structure (Histone 1 (H1), Histone 2a (H2A), Histone 2b (H2B), Histone 3 (H3), Histone 4 (H4)) was lower in the testes of 3nBY, whereas the expression of genes encoding ubiquitin (Ubiquitin Conjugating Enzymes (UBEs), Ring Finger Proteins (RNFs)) and apoptosis (CASPs (Caspase 3, Caspase 7,Caspase 8), BCLs (B-Cell Lymphoma 3, B-Cell CLL/Lymphoma 2, B Cell CLL/Lymphoma 10)) proteins involved in spermatid degeneration was higher. These data suggest that the disrupted expression of genes associated with spermatogenesis and the increased expression of mitochondrial ubiquitin, which initiates cell apoptosis, may result in spermatid degeneration in male 3nBY. This study provides information regarding the potential molecular regulatory mechanisms underlying male infertility in polyploid fish.

Asymmetric expression patterns reveal a strong maternal effect and dosage compensation in polyploid hybrid fish.

BACKGROUND: Hybridization and polyploidization are regarded as the major driving forces in plant speciation, diversification, and ecological adaptation. Our knowledge regarding the mechanisms of duplicated-gene regulation following genomic merging or doubling is primarily derived from plants and is sparse for vertebrates. RESULTS: We successfully obtained an F1 generation (including allodiploid hybrids and triploid hybrids) from female Megalobrama amblycephala Yih (BSB, 2n = 48) × male Xenocypri davidi Bleeker (YB, 2n = 48). The duplicated-gene expression patterns of the two types of hybrids were explored using RNA-Seq data. In total, 5.44 × 108 (69.32 GB) clean reads and 499,631 assembled unigenes were obtained from the testis transcriptomes. The sequence similarity analysis of 4265 orthologs revealed that the merged genomes were dominantly expressed in different ploidy hybrids. The differentially expressed genes in the two types of hybrids were asymmetric compared with those in both parents. Furthermore, the genome-wide expression level dominance (ELD) was biased toward the maternal BSB genome in both the allodiploid and triploid hybrids. In addition, the dosage-compensation mechanisms that reduced the triploid expression levels to the diploid state were determined in the triploid hybrids. CONCLUSIONS: Our results indicate that divergent genomes undergo strong interactions and domination in allopolyploid offspring. Genomic merger has a greater effect on the gene-expression patterns than genomic doubling. The various expression mechanisms (including maternal effect and dosage compensation) in different ploidy hybrids suggest that the initial genomic merger and doubling play important roles in polyploidy adaptation and evolution.

Determination of dosage compensation and comparison of gene expression in a triploid hybrid fish.

BACKGROUND: Polyploidy and hybridization are both recognized as major forces in evolution. Most of our current knowledge about differences in gene regulation in polyploid hybrids comes from plant studies. The gene expression of diverged genomes and regulatory interactions are still unclear in lower vertebrates. RESULTS: We generated 229 million cleaned reads (42.23 Gbp) from triploid of maternal grass carp (Ctenopharyngodon idellus, Cyprininae, 2n = 48) × paternal blunt snout bream (Megalobrama amblycephala, Cultrinae, 2n = 48) and their diploid parents using next-generation sequencing. In total, 157,878 contigs were assembled and 15,444 genes were annotated. We examined gene expression level changes among the parents and their triploid offspring. The mechanisms of dosage compensation that reduced triploid expression levels to the diploid state were determined in triploid fish. In this situation, novel gene expression and gene silencing were observed. Then, we established a model to determine the extent and direction of expression level dominance (ELD) and homoeolog expression bias (HEB) based on the relative expression level among the parents and their triploid offspring. CONCLUSIONS: Our results showed that the genome-wide ELD was biased toward maternal genome in triploid. Extensive alterations in homoeolog expression suggested a combination of regulatory and epigenetic interactions through the transcriptome network. Additionally, the expression patterns of growth genes provided insights into the relationship between the characteristics of growth and underlying mechanisms in triploids. Regulation patterns of triploid state suggest that various expression levels from the initial genomic merger have important roles in adaptation.

Unique nucleolar dominance patterns in distant hybrid lineage derived from Megalobrama Amblycephala × Culter Alburnus.

BACKGROUND: Nucleolar dominance is an epigenetic phenomenon that occurs in interspecific hybrids and involves the expression of 45S rRNA genes inherited from one progenitor due to the silencing of the other progenitor's rRNA genes. In this paper, changes in the genetics and expression of 45S rRNA genes in F1 and F2 hybrid progeny of blunt snout bream (BSB, Megalobrama amblycephala) × topmouth culter (TC, Culter alburnus) are investigated. RESULTS: The 45S rDNA loci were analyzed by cloning, RT-PCR and sequencing methods. The results show that nucleolar dominance patterns differ in the F1 and F2 hybrids. In the F1 hybrids of BSB × TC, all the tested individuals inherited and expressed the 45S rRNA genes of both BSB and TC, indicating that nucleolar dominance is not established in the F1 hybrids. However, in the F2 hybrids of BSB × TC, five patterns are observed. Pattern 1 inherits and expresses only the 45S rRNA gene of BSB. Pattern 2 inherits the 45S rRNA gene from both BSB and TC, but only expresses the 45S rRNA of BSB. Pattern 3 inherits and expresses the 45S rRNA gene from both BSB and TC. Pattern 4 inherits the 45S rRNA gene from both BSB and TC, but only expresses the 45S rRNA gene of TC. Pattern 5 inherits and expresses only the 45S rRNA gene of TC. CONCLUSIONS: Nucleolar dominance shows distinctive patterns in intergeneric hybrids of BSB × TC. It is not established in F1 hybrids and is random in F2 hybrids. This study provides new insights into the phenomenon of nucleolar dominance in genetic hybrids in vertebrates.

Effects of Ti Target Purity and Microstructure on Deposition Rate, Microstructure and Properties of Ti Films.

Three titanium (Ti) targets with different purities were used to prepare Ti films on polyimide substrates by DC magnetron sputtering. The microstructures of Ti films were characterized by a metallographic microscope, X-ray diffractometer, field emission scanning electron microscope and three-dimensional surface topography instrument. In this study, we investigated the effects of Ti target purity and microstructure on film deposition rate, surface roughness, microstructure and resistivity. The results show that the deposition rate increased with increasing Ti target purity. Ti film deposited by the high-purity (99.999%) Ti target has fewer surface particles with smaller size, lower surface roughness and lower resistivity when compared to that prepared by the Ti target of low purity (99.7%). The surface roughness of Ti film prepared by the high-purity Ti target was Sa = 121 nm, the deposition rate was 16.3 nm/min and the resistivity was 6.9 × 10-6 Ω·m. For Ti targets of the same purity, the performance of Ti film prepared by a target with equiaxed α-phase grains is better than that of Ti film prepared by a target with twins and ß-phase grains.

Comparative Analysis of the Gut Microbiota of Mandarin Fish (Siniperca chuatsi) Feeding on Compound Diets and Live Baits.

Siniperca chuatsi feeds on live fry throughout their life. The sustainable development of its farming industry has urgently necessitated the development of artificial diets to substitute live baits. It has been demonstrated that gut microbiota assists in feed adaptation and improves the feed conversion rate in fish. Therefore, this study aimed to understand the potential role of intestinal microorganisms in the domestication of S. chuatsi with a compound diet. Accordingly, we performed 16S rRNA sequencing of the gut microbial communities in S. chuatsi groups that were fed a compound diet (including large and small individuals) and live baits. A total of 2,471 OTUs were identified, and the large individual group possessed the highest number of unique OTUs. The α-diversity index of the gut microbiota in groups that were fed a compound diet was significantly higher (p < 0.05) than that in the live bait group. There were no significant differences in the α-diversity between the large and small individual groups. However, relatively higher numbers of Lactococcus, Klebsiella, and Woeseia were observed in the intestines of the large individual group. Prediction of the metabolic function of the microbiota among these three fish groups by Tax4Fun revealed that most metabolic pathways, such as glycan metabolism and amino acid metabolism, were typically more enriched for the larger individuals. The results indicated that certain taxa mentioned above exist in large individuals and may be closely related to the digestion and absorption of compound diets. The present study provides a basis for understanding the utilization mechanism of artificial feed by S. chuatsi.

Exposure to heat stress causes downregulation of immune response genes and weakens the disease resistance of Micropterus salmoides.

In order to understand the molecular mechanism of response to heat stress in largemouth bass (LMB) Micropterus salmoides, we performed transcriptome analysis of spleen tissue of LMB subjected to heat stress and challenged with A. veronii under heat stress. A total of 2162 DEGs were identified between the heat stressed (32 °C) and control groups (24 °C) after 7 d treatment. Gene Ontology (GO) annotation analysis revealed that these differentially expressed genes (DEGs) were mainly enriched on GO terms of biological regulation, membrane part, and binding. ELISA validation indicated that except major histocompatibility complex II (Mhc II), the protein levels of t-Sod, caspase 3 (Casp3), tumor necrosis factor-α (Tnf-α), and complement component 3 (C3) were consistent with RNA-seq results. In the experiment of A. veronii challenged under heat stress (32 °C), 2899 and 2663 DEGs were obtained from the heat stress-challenged group (H6 vs H0, H12 vs H0), while 1485 and 3501 DEGs from the control-challenged group (C6 vs C0, C12 vs C0). GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that immune-related categories and pathways were significantly enriched, such as immune system process, immune response and positive regulation of immune response in GO enrichment analysis, and cytokine-cytokine receptor interaction, human cytomegalovirus infection in KEGG signaling pathways. The expressions of f11, c1q and c3 in complement and coagulation pathway, as well as that of proinflammatory genes tnf-α and il-8, were deeply inhibited. Real-time quantitative PCR validation for nine DEGs showed that most of them had consistent expression trends with RNA-seq results. Our results indicated that heat stress affects the immunity and metabolism of LMB. In particular, it aggravates the inhibitory effects of A. veronii on the complement and coagulation systems while downregulating proinflammatory cytokine expression, thereby weakening the resistance of LMB to pathogen infection. Our results contribute to the elucidation of A. veronii infection pathogenic mechanisms in LMB under heat stress.

Characterization of kiss2/kissr2 system in largemouth bass (Micropterus salmoides) and Kiss2-10 peptide regulation of the hypothalamic-pituitary-gonadal axis.

The kisspeptin system, which lies upstream of the hypothalamic-pituitary-gonadal (HPG) axis, is believed to function as a regulator of reproduction in teleosts. In this study, we isolated and characterized kiss2 and its receptor kissr2 in largemouth bass (Micropterus salmoides). The complete coding sequences of kiss2 and kissr2 were 375 and 1134 bp long and encoded precursor proteins 124 and 377 amino acid long, respectively. Real-time PCR showed that kiss2 and kissr2 were primarily expressed in the HPG axis. The expression profile of kiss2 and kissr2 varied with gonadal development, with the highest and lowest expression levels being detected during the immature and final maturation stages, respectively. Intraperitoneal injection of exogenous Kiss2-10 peptide increased the transcript levels of gnrh3, kissr2, fshß, lhß, ar, and er2 within 24 h (p < 0.05), as well as plasma levels of 17ß-estradiol and testosterone. Histological analysis indicated that chronic administration of exogenous Kiss2-10 peptide accelerated vitellogenesis in females and spermatogenesis in males. Further, in situ hybridization revealed that kiss2 is expressed in the ooplasm and vitelline envelope of oocytes and the spermatocytes of testes. In addition, experiments using gonad tissue primary cell cultures indicated that exogenous Kiss2-10 peptide stimulates the expression of reproduction-related genes. Collectively, our findings indicate that the kiss2/kissr2 system in largemouth bass is involved in regulating gonadal development through the HPG axis.

Chromosome-level genome assembly for the largemouth bass Micropterus salmoides provides insights into adaptation to fresh and brackish water.

Largemouth bass (LMB; Micropterus salmoides) has been an economically important fish in North America, Europe, and China. This study obtained a chromosome-level genome assembly of LMB using PacBio and Hi-C sequencing. The final assembled genome is 964 Mb, with contig N50 and scaffold N50 values of 1.23 Mb and 36.48 Mb, respectively. Combining with RNA sequencing data, we annotated a total of 23,701 genes. Chromosomal assembly and syntenic analysis proved that, unlike most Perciformes with the popular haploid chromosome number of 24, LMB has only 23 chromosomes (Chr), among which the Chr1 seems to be resulted from a chromosomal fusion event. LMB is phylogenetically closely related to European seabass and spotted seabass, diverging 64.1 million years ago (mya) from the two seabass species. Eight gene families comprising 294 genes associated with ionic regulation were identified through positive selection, transcriptome and genome comparisons. These genes involved in iron facilitated diffusion (such as claudin, aquaporins, sodium channel protein and so on) and others related to ion active transport (such as sodium/potassium-transporting ATPase and sodium/calcium exchanger). The claudin gene family, which is critical for regulating cell tight junctions and osmotic homeostasis, showed a significant expansion in LMB with 27 family members and 68 copies for salinity adaptation. In summary, we reported the first high-quality LMB genome, and provided insights into the molecular mechanisms of LMB adaptation to fresh and brackish water. The chromosome-level LMB genome will also be a valuable genomic resource for in-depth biological and evolutionary studies, germplasm conservation and genetic breeding of LMB.

Comparative Genomics Studies on the dmrt Gene Family in Fish.

Doublesex and mab-3-related transcription factor (dmrt) genes are widely distributed across various biological groups and play critical roles in sex determination and neural development. Here, we applied bioinformatics methods to exam cross-species changes in the dmrt family members and evolutionary relationships of the dmrt genes based on genomes of 17 fish species. All the examined fish species have dmrt1-5 while only five species contained dmrt6. Most fish harbored two dmrt2 paralogs (dmrt2a and dmrt2b), with dmrt2b being unique to fish. In the phylogenetic tree, 147 DMRT are categorized into eight groups (DMRT1-DMRT8) and then clustered in three main groups. Selective evolutionary pressure analysis indicated purifying selections on dmrt1-3 genes and the dmrt1-3-2(2a) gene cluster. Similar genomic conservation patterns of the dmrt1-dmrt3-dmrt2(2a) gene cluster with 20-kb upstream/downstream regions in fish with various sex-determination systems were observed except for three regions with remarkable diversity. Synteny analysis revealed that dmrt1, dmrt2a, dmrt2b, and dmrt3-5 were relatively conserved in fish during the evolutionary process. While dmrt6 was lost in most species during evolution. The high conservation of the dmrt1-dmrt3-dmrt2(2a) gene cluster in various fish genomes suggests their crucial biological functions while various dmrt family members and sequences across fish species suggest different biological roles during evolution. This study provides a molecular basis for fish dmrt functional analysis and may serve as a reference for in-depth phylogenomics.

Evidence for the paternal mitochondrial DNA in the crucian carp-like fish lineage with hybrid origin.

In terms of taxonomic status, common carp (Cyprinus carpio, Cyprininae) and crucian carp (Carassius auratus, Cyprininae) are different species; however, in this study, a newborn homodiploid crucian carp-like fish (2n=100) (2nNCRC) lineage (F1-F3) was established from the interspecific hybridization of female common carp (2n=100)×male blunt snout bream (Megalobrama amblycephala, Cultrinae, 2n=48). The phenotypes and genotypes of 2nNCRC differed from those of its parents but were closely related to those of the existing diploid crucian carp. We further sequenced the whole mitochondrial (mt) genomes of the 2nNCRC lineage from F1 to F3. The paternal mtDNA fragments were stably embedded in the mt-genomes of F1-F3 generations of 2nNCRC to form chimeric DNA fragments. Along with this chimeric process, numerous base sites of F1-F3 generations of 2nNCRC underwent mutations. Most of these mutation sites were consistent with the existing diploid crucian carp. Moreover, the mtDNA organization and nucleotide composition of 2nNCRC were more similar to those of the existing diploid crucian carp than those of the parents. The inheritable chimeric DNA fragments and mutant loci in the mt-genomes of different generations of 2nNCRC provided important evidence of the mtDNA change process in the newborn lineage derived from hybridization of different species. Our findings demonstrated for the first time that the paternal mtDNA were transmitted into the mt-genomes of homodiploid lineage, which provided new insights into the existence of paternal mtDNA in the mtDNA inheritance.

Blue, green, and red full-color ultralong afterglow in nitrogen-doped carbon dots.

Carbon dots (CDs) with tunable emission colors and multiple emission modes are highly desirable in advanced optical anti-counterfeiting. Some pioneering efforts to trigger additional long-lived emission modes, nevertheless, did not perfectly solve the issue of printability and color-tunability in practical applications. Herein, we developed an encapsulating-dissolving-recrystallization route for the synthesis of CD-based anti-counterfeiting inks, and accordingly realized blue, green, and red full-color afterglow emissions from these CD-based inks when printed on paper. The printed inks simultaneously possessed triple emission modes including fluorescence (FL), delayed fluorescence (DF), and room-temperature phosphorescence (RTP), among which the long-lived emissions (DF and RTP) could be selectively activated by using different excitation wavelengths. We believe that the proposed synthetic route in this work may promote the development of multicolor-encoded and multiple-mode-integrated optical anti-counterfeiting systems, and will expand the application of CD-based materials to the fields of sensing, photodynamic therapy and bio-imaging.

Transcriptome Profile Analysis on Ovarian Tissues of Autotetraploid Fish and Diploid Red Crucian Carp.

Polyploidization can significantly alter the size of animal gametes. Autotetraploid fish (RRRR, 4nRR = 200) (4nRR) possessing four sets of chromosomes were derived from whole-genome duplication in red crucian carp (RR, 2n = 100) (RCC). The diploid eggs of the 4nRR fish were significantly larger than the eggs of RCC. To explore the differences between the ovaries of these two ploidies of fishes at the molecular level, we compared the ovary transcriptome profiles of 4nRR fish and RCC and identified differentially expressed genes (DEGs). A total of 19,015 unigenes were differentially expressed between 4nRR fish and RCC, including 12,591 upregulated and 6,424 downregulated unigenes in 4nRR fish. Functional analyses revealed that eight genes (CDKL1, AHCY, ARHGEF3, TGFß, WNT11, CYP27A, GDF7, and CKB) were involved in the regulation of cell proliferation, cell division, gene transcription, ovary development and energy metabolism, suggesting that these eight genes were related to egg size in 4nRR fish and RCC. We validated the expression levels of these eight DEGs in 4nRR fish and RCC using quantitative PCR. The study results provided insights into the regulatory mechanisms underlying the differences in crucian carp egg sizes.

Targeted disruption of tyrosinase causes melanin reduction in Carassius auratus cuvieri and its hybrid progeny.

The white crucian carp (Carassius auratus cuvieri, WCC) not only is one of the most economically important fish in Asia, characterized by strong reproductive ability and rapid growth rates, but also represents a good germplasm to produce hybrid progenies with heterosis. Gene knockout technique provides a safe and acceptant way for fish breeding. Achieving gene knockout in WCC and its hybrid progeny will be of great importance for both genetic studies and hybridization breeding. Tyrosinase (TYR) is a key enzyme in melanin synthesis. Depletion of tyr in zebrafish and mice results in mosaic pigmentation or total albinism. Here, we successfully used CRISPR-Cas9 to target tyr in WCC and its hybrid progeny (WR) derived from the cross of WCC (♀) and red crucian carp (Carassius auratus red var., RCC, ♂). The level of TYR protein was significantly reduced in mutant WCC. Both the mutant WCC and the mutant WR showed different degrees of melanin reduction compared with the wild-type sibling control fish, resulting from different mutation efficiency ranging from 60% to 90%. In addition, the transcriptional expression profiles of a series of pivotal pigment synthesis genes, i.e. tyrp1, mitfa, mitfb, dct and sox10, were down-regulated in tyr-CRISPR WCC, which ultimately caused a reduction in melanin synthesis. These results demonstrated that tyr plays a key role in melanin synthesis in WCC and WR, and CRISPR-Cas9 is an effective tool for modifying the genome of economical fish. Furthermore, the tyr-CRISPR models could be valuable in understanding fundamental mechanisms of pigment formation in non-model fish.

The Sterility of Allotriploid Fish and Fertility of Female Autotriploid Fish.

Based on the formation of an autotetraploid fish line (4nAUT, 4n = 200; F2-F11) derived from the distant hybridization of female Carassius auratus red var. (RCC, 2n = 100) × male Megalobrama amblycephala (BSB, 2n = 48), we produced autotriploid hybrids (3nAUT) by crossing females of RCC with males of 4nAUT and allotriploid hybrids (3nALT) by crossing females of Cyprinus carpio (CC, 2n = 100) with males of 4nAUT. The aim of this study was to comparatively investigate the reproductive characteristics of 3nALT and 3nAUT. We investigated morphological traits, chromosomal numbers, DNA content and gonadal development in 3nAUT and 3nALT. The results indicated both 3nAUT and 3nALT possessed 150 chromosomes and were triploid hybrids. The females and males of 3nALT and males of 3nAUT had abnormal gonadal development and could not generate mature eggs or sperm, but the females of 3nAUT had normal gonadal development and generated mature eggs at 2 years old. The females of 3nAUT generated different sizes of eggs, which fertilized with haploid sperm from RCC and formed viable diploid, triploid, and tetraploid offspring. The formation of these two kinds of triploid hybrids provides an ideal model for studying the reproductive traits of triploid hybrids, which is of great value in animal genetics and reproductive biology.