Identification and effective regulation of scarb1 gene involved in pigmentation change in autotetraploid Carassius auratus

The autotetraploid Carassius auratus(4nRR,4n=200,RRRR)is derived from whole-genome duplication of Carassius auratus red var.(RCC,2n=100,RR).In the current study,we demonstrated that chromatophores and pigment changes directly caused the coloration and variation of 4nRR skin(red in RCC,brownish-yellow in4nRR).To further explore the molecular mechanisms underlying coloration formation and variation in 4nRR,we performed transcriptome profiling and molecular functional verification in RCC and 4nRR.Results revealed that scarb1,associated with carotenoid metabolism,underwent significant down-regulation in 4nRR.Efficient editing of this candidate pigment gene provided clear evidence of its significant role in RCC coloration.Subsequently,we identified four divergent scarb1 homeologs in 4nRR:two original scarb1 homeologs from RCC and two duplicated ones.Notably,three of these homeologs possessed two highly conserved alleles,exhibiting biased and allelespecific expression in the skin.Remarkably,after precise editing of both the original and duplicated scarb1homeologs and/or alleles,4nRR individuals,whether singly or multiply mutated,displayed a transition from brownishyellow skin to a cyan-gray phenotype.Concurrently,the proportional areas of the cyan-gray regions displayed a gene-dose correlation.These findings illustrate the subfunctionalization of duplicated scarb1,with all scarb1genes synergistically and equally contributing to the pigmentation of 4nRR.This is the first report concerning the functional differentiation of duplicated homeologs in an autopolyploidfish,substantiallyenrichingour understanding of coloration formation and change within this group of organisms.

The key role of myostatin b in somatic growth in fishes derived from distant hybridization

The basic mechanism of heterosis has not been systematically and completely characterized.In previous studies,we obtained three economically important fishes that exhibit rapid growth,WR(WCC♀×RCC♂),WR-Ⅱ(WR♀×WCC♂),and WR-Ⅲ(WR-Ⅱ♀×4nAU♂),through distant hybridization.However,the mechanism underlying this rapid growth remains unclear.In this study,we found that WR,WR-Ⅱ,and WR-Ⅲshowed muscle hypertrophy and higher muscle protein and fat contents compared with their parent species(RCC and WCC).Candidate genes responsible for this rapid growth were then obtained through an analysis of 12 muscle transcriptomes.Notably,the mRNA level of mstnb(myostatin b),which is a negative regulator of myogenesis,was significantly reduced in WR,WR-Ⅱ,and WR-Ⅲcompared with the parent species.To verify the function of mstnb,a mstnb-deficient mutant RCC line was generated using the CRISPR-Cas9 technique.The average body weight of mstnb-deficient RCC at 12 months of age was significantly increased by 29.57%compared with that in wild-type siblings.Moreover,the area and number of muscle fibers were significantly increased in mstnb-deficient RCC,indicating hypertrophy and hyperplasia.Furthermore,the muscle protein and fat contents were significantly increased in mstnb-deficient RCC.The molecular regulatory mechanism of mstnb was then revealed by transcription profiling,which showed that genes related to myogenesis(myod,myog,and myf5),protein synthesis(PI3K-AKT-mTOR),and lipogenesis(pparγand fabp3)were highly activated in hybrid fishes and mstnb-deficient RCC.This study revealed that low expression or deficiency of mstnb regulates somatic growth by promoting myogenesis,protein synthesis,and lipogenesis in hybrid fishes and mstnb-deficient RCC,which provides evidence for the molecular mechanism of heterosis via distant hybridization.

Ccdc57 is required for straightening the body axis by regulating ciliary motility in the brain ventricle of zebrafish

Recently,cilia defects have been proposed to contribute to scoliosis.Here,we demonstrate that coiled-coil domain-containing 57(Ccdc57)plays an essential role in straightening the body axis of zebrafish by regulating ciliary beating in the brain ventricle(BV).Zygotic ccdc57(Zccdc57)mutant zebrafish developes scoliosis without significant changes in their bone density and calcification,and the maternal-zygotic ccdc57(MZccdc57)mutant embryos display curved bodies since the long-pec stage.The expression of ccdc57 is enriched in ciliated tissues and immunofluorescence analysis reveals colocalization of Ccdc57-HA with acetylated a-tubulin,implicating it in having a role in ciliary function.Further examination reveals that it is the coordinated cilia beating of multiple cilia bundles(MCB)in the MZccdc57 mutant embryos that is affected at 48 hours post fertilization,when the compromised cerebrospinal fluid flow and curved body axis have already occurred.Either ccdc57 m RNA injection or epinephrine treatment reverses the spinal curvature in MZccdc57 mutant larvae from ventrally curly to straight or even dorsally curly and significantly upregulates urotensin signaling.This study reveals the role of ccdc57 in maintaining coordinated cilia beating of MCB in the BV.

Establishment and application of distant hybridization technology in fish

Hybridization is widely used. However, for a long time, systematic theories and technologies related to hybridization in fish have been lacking. In this study, through long-term systematic research, we investigated and obtained the main rules regarding inheritance and reproduction related to fish distant hybridization. Furthermore, we established one-step and multistep breeding technologies that were suitable for interspecific hybridization and intraspecific hybridization. Simultaneously, we used these two breeding technologies to produce a batch of diploid fish lineages and tetraploid fish lineages and improved fishes. In addition, we widely discuss the methods, technologies and results of hybridization breeding, referring to the domestic and foreign literature on fish hybridization. We hope that this paper will be beneficial for the research and application of fish hybrid breeding.

Characterization of H3 methylation in regulating oocyte development in cyprinid fish

Histone post-modifications are important epigenetic markers involved in multiple cellular processes via regulation of gene transcription or remodeling of chromatin structure. Oocyte development is a critical process under rigorous control to prevent the generation of aberrant gametes. However, the regulatory mechanism of oocyte early development is not well-understood due to the tiny size and poor distinguishability of the gonad in juvenile stages. Here, two cyprinid hybrid fishes, a sterile allotriploid fish and a gynogenetic hybrid fish with delayed oocyte development, provided research models to investigate the mechanisms involved. We used cytogenetic and molecular methods to confirm the pachytene arrest of oocytes in allotriploid fish and gynogenetic hybrid fish. On the basis of these developmental differences, we screened 21 different histone H3 modifications by ELISA and found that four modifications(H3 K4 me3, H3 K9 me3, H3 K79 me, and H3 K79 me3) differed significantly in the two cyprinid hybrid fishes. Changes in histone methylation at the three residues(H3 K4, K9, K79) were caused by specific methyltransferases and demethylases. Our results provide new insights into the epigenetic regulation of oocyte early development in fish, a process critical for understanding of reproductive biology and with practical applications in the aquacultural breeding industry.

Diverse transcriptional patterns of homoeologous recombinant transcripts in triploid fish(Cyprinidae)

Homoeologous recombination(HR),the exchange of homoeologous chromosomes,contributes to subgenome adaptation to diverse environments by producing various phenotypes.However,the potential relevance of HR and innate immunity is rarely described in triploid cyprinid fish species.In our study,two allotriploid genotypes(R_(2)C and RC_(2)),whose innate immunity was stronger than their inbred parents(Carassius auratus red var.and Cyprinus carpio L.),were obtained from backcrossing between male allotetraploids of C.auratus red var.×C.carpio L.and females of their two inbred parents,respectively.The work detected 140 HRs shared between the two triploids at the genomic level.Further,transcriptions of 54 homoeologous recombinant genes(HRGs)in R_(2)C and 65 HRGs in RC_(2) were detected using both Illumina and PacBio data.Finally,by comparing expressed recombinant reads to total expressed reads in each of the genes,a range of 0.1%-10% was observed in most of the 99-193 HRGs,of which six recombinant genes were classified as"response to stimulus".These results not only provide a novel way to predict HRs in allopolyploids based on cross prediction at both genomic and transcriptional levels,but also insight into the potential relationship between HRs related to innate immunity and adaptation of the triploids and allotetraploids.

The chimeric genes in the hybrid lineage of Carassius auratus cuvieri(♀)×Carassius auratus red var.(♂)

Hybridization can combine the genomes of different strains or species, which leads to changes of genotype and phenotype in the hybrids. In this study, we aimed to investigate the genetic variations of hybrids(WR-F1 and WR-F2) derived from the intraspecific hybridization of white crucian carp(Carassius auratus cuvieri, WCC, ♀) and red crucian carp(Carassius auratus red var., RCC, ♂). Here, we compared the orthologous genes in the liver transcriptomes of hybrids with those of WCC and RCC, and classified the orthologous genes into eight gene patterns within three categories(chimera, mutant, and biparental origin genes).The results revealed 19.04%, 4.17% chimeric genes and 6.90%, 5.05% mutations of orthologous genes in WR-F1 and WR-F2 respectively. Seventeen of twenty-three characterized genes(77%) were confirmed to be the chimeras at the genomic DNA level.The GO classification discovered that some chimeric and mutant genes were related to metabolic process, immune system and developmental process in WR-F1. Our results provide the new evidence that hybridization can combine the parental genomes,leading to changes in the genotype of the resultant hybrids. This is the first report on the formation of chimeric genes from fish intraspecific hybridization, which is potentially interesting from the context of both evolution and the genetic breeding of fish.

A comparative study of distant hybridization in plants and animals

Distant hybridization refers to crosses between two different species, genera, or higher-ranking taxa, which can break species limits, increase genetic variation, and combine the biological characteristics of existing species. It is an important way of creating genetic variation, fertile strains, and excellent characteristics in new strains and populations. Combining analyses and summaries from many inter-related documents in plants and animals, both domestic and international, including examples and long-standing research on distant hybridization in fish from our laboratory, we summarize and compare the similarities and differences in plant and animal distant hybridization. In addition, we analyze and review the biological characteristics of their different ploidy progenies and the possible causes of disparity in survival rates. Mechanisms of sterility in animal and plant distant hybrids are also discussed, and research methods for the study of biological characteristics of hybrids, including morphology, cytology,and molecular cytogenetics are presented. This paper aims to provide comprehensive research materials and to systematically compare the general and specific characteristics of plant and animal hybrids with regards to reproduction, genetics, growth traits,and other biological characteristics. It is hoped that this paper will have great theoretical and practical significance for the study of genetic breeding and biological evolution of plant and animal distant hybridization.

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.

Comparison of diploid and triploid Carassius auratus provides insights into adaptation to environmental change

Focusing on adaptation of aquatic organisms, especially fish, can help elucidate complex dynamics in freshwater ecology. The differences in genetic and epigenetic regulation between diploid and triploid Carassius auratus affect survival under eutrophication. To identify the underlying mechanisms that lead to better adaption of triploids than diploids, we compared mRNA and microRNA(miRNA) expressions in liver tissue of diploid and triploid individuals obtained from the Dongting lake water system in central China. Differential expression analysis revealed that 566 transcripts were significantly up-regulated, whereas758 were down-regulated in triploids; of these differentially expressed transcripts, 33 transcripts including cacna1 d, nfkb2, hspa1 and fgfr4 were involved in the MAPK signaling pathway, and eight transcripts were determined to be regulated by seven miRNAs. Additionally, four of 25 differential expressed(DE) transcripts(mhc1, irf7, nfkb2 and pik3 c) involving the viral carcinogenesis pathway were regulated by four miRNAs. Furthermore, genetic polymorphisms analysis showed that more heterozygous mutations were detected in triploids than diploids. The d N/d S results revealed that 21 genes were under positive selection(d N/d S>1) in C. auratus complex. We hypothesize that these changes related to genetic and epigenetic regulation may be caused by abiotic stresses, and facilitate adaptation to environmental changes.

Assess Medical Screening and Isolation Measures Based on Numerical Method for COVID-19 Epidemic Model in Japan

This study aims to improve control schemes for COVID-19 by a numerical model with estimation of parameters.We established a multi-level and multi-objective nonlinear SEIDR model to simulate the virus transmission.The early spread in Japan was adopted as a case study.The first 96 days since the infection were divided into five stages with parameters estimated.Then,we analyzed the trend of the parameter value,age structure ratio,and the defined PCR test index(standardization of the scale of PCR tests).It was discovered that the self-healing rate and confirmed rate were linear with the age structure ratio and the PCR test index using the stepwise regression method.The transmission rates were related to the age structure ratio,PCR test index,and isolation efficiency.Both isolation measures and PCR test medical screening can effectively reduce the number of infected cases based on the simulation results.However,the strategy of increasing PCR test medical screening would encountered a bottleneck effect on the virus control when the index reached 0.3.The effectiveness of the policy would decrease and the basic reproduction number reached the extreme value at 0.6.This study gave a feasible combination for isolation and PCR test by simulation.The isolation intensity could be adjusted to compensate the insufficiency of PCR test to control the pandemic.

miR-199-5p regulates spermiogenesis at the posttranscriptional level via targeting Tekt1 in allotriploid crucian carp

Background:Sperm abnormalities are one of the primary factors leading to male sterility,but their pathogenesis is still unclear.Although miRNAs are suggested to exert important roles in the regulation of spermatogenesis at both transcriptional and posttranscriptional levels,little is currently known regarding the regulation of sperm flagella assembly by microRNAs(miRNAs).The role of miRNAs in the development of sperm abnormalities in sterile triploid fish has not been studied.Results:In this study,we found that miR-199-5p was widely expressed in all detected tissues of different-ploidy crucian carp.As one of the testis-specific candidate markers,Tekt1 was predominantly expressed in the testis.Quantitative real-time PCR(qRT-PCR)analyses showed that the expression trend of miR-199-5p was exactly opposite to that of Tekt1.Through bioinformatics analysis,we identified a putative miR-199-5p binding site in the Tekt1 mRNA.We further identified Tekt1 as a target of miR-199-5p using luciferase reporter assay.Finally,we confirmed that miR-199-5p was necessary for sperm flagellar assembly and spermatogenesis in vivo via intraperitoneal injection of miR-199-5p antagomir or agomir in diploid red crucian carp.Moreover,miR-199-5p gainof-function could lead to spermatids apoptosis and abnormal spermatozoa structure,which is similar to that of allotriploid crucian carp.Conclusions:Our studies suggested that abnormally elevated miR-199-5p inhibited the sperm flagella formation in spermiogenesis by negatively regulating the expression of Tekt1,thereby causing sperm abnormalities of male allotriploid crucian carp.

An improved hybrid bream derived from a hybrid lineage of Megalobrama amblycephala(♀)×Culter alburnus(♂)

Distant hybridization is an important technique in fish genetic breeding.In this study,based on the establishment of an allodiploid fish lineage(BT,2n=48,F_(1)–F_(6))derived from distant hybridization between female Megalobrama amblycephala(BSB,2n=48)and male Culter alburnus(TC,2n=48),and the backcross progeny(BTB,2n=48)derived by backcrossing female F_(1) of BT to male BSB,an improved hybrid bream(BTBB,2n=48)was obtained by backcrossing BTB(♀)to BSB(♂).Moreover,the morphological and genetic characteristics of BTBB individuals were investigated;BTBB was similar to BSB in appearance but had a higher body height than BSB.The study results regarding chromosome numbers and DNA content indicated that BTBB is a diploid hybrid fish.The 5S rDNA and Hox gene of BTBB were inherited from the original parents.Gonadal development in BTBB was normal.On the other hand,BTBB had a faster growth rate,higher muscle protein level,and lower muscle carbohydrate level than BSB.Hence,bisexual fertile BTBB is promoted and can be applied as a high-quality fish,and it can also be used as a new fish germplasm resource to develop high-quality fish further.Thus,this study is of great significance for fish genetic breeding.

Estimating SVCV waterborne transmission and predicting experimental epidemic development:A modeling study using a machine learning approach

Viral infectious diseases significantly threaten the sustainability of freshwater fish aquaculture.The lack of studies on epidemic transmission patterns and mechanisms inhibits the development of containment strategies from the viewpoint of veterinary public health.This study raises an epidemic mathematical model considering water transmission with the aim of analyzing the transmission process more accurately.The basic reproduction number R0 was derived by the model parameter including the water transmission coefficient and was used for the analysis of the virus transmission.Spring viremia of carp virus(SVCV)and zebrafish were used as model viruses and animals,respectively,to conduct the transmission experiment.Transmission through water was achieved by connecting two aquarium tanks with a water channel but blocking the fish movement between the tanks.With the collected experimental data,we determined the optimal hybrid machine learning algorithm to analyze the transmission process using an established mathematical model.In addition,future transmission was predicted and validated using the epidemic model and an optimal algorithm.Finally,the sensitivity of model parameters and the simulations of R0 variation were performed based on the modified complex epidemic model.This study is of significance in providing theoretical guidance for minimizing R0 by manipulating model parameters with containment measures.More importantly,since the modified model and algorithm demonstrated better performance in handling freshwater fish transmission problems,this study advances the future application of transmissible disease modeling with larger datasets in freshwater fish aquaculture.

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 2 n NCRC 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 2n NCRC lineage from F1 to F3.The paternal mt DNA fragments were stably embedded in the mt-genomes of F1–F3 generations of 2n NCRC to form chimeric DNA fragments.Along with this chimeric process,numerous base sites of F1–F3 generations of 2 n NCRC underwent mutations.Most of these mutation sites were consistent with the existing diploid crucian carp.Moreover,the mt DNA organization and nucleotide composition of 2n NCRC 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 mt DNA change process in the newborn lineage derived from hybridization of different species.Our findings demonstrated for the first time that the paternal mt DNA were transmitted into the mt-genomes of homodiploid lineage,which provided new insights into the existence of paternal mt DNA in the mt DNA inheritance.

Abnormal meiosis in fertile and sterile triploid cyprinid fish

Meiosis is the key process for producing mature gametes. A natural fertile triploid Carassius auratus population(3nDTCC) and an artificially derived sterile triploid crucian carp(3nCC) have been previously observed, providing suitable model organisms for investigating meiosis characteristics in triploid fish. In the present study, the microstructures and ultrastructures of spermatogenesis were studied in these fishes. Td T-mediated d UTP nick end labeling detection was performed to investigate the apoptosis of spermatocytes. Fluorescence in situ hybridization was employed to trace chromatin pairing. In addition, the m RNA expressions of cell cycle-related genes(i.e., cell division control 2 and cell cycle protein B) were determined by quantitative realtime polymerase chain reaction to illustrate the molecular mechanism of abnormal meiosis in the 3nCC. The results showed that the 3nCC undergoes an irregular prophase I, with the chromosomes distributed in a unipolar radial manner and exhibiting partial pairing, hindered metaphase I, and degenerated cells in the subsequent stages. Meanwhile, the 3nDTCC presented a relatively regular meiotic prophase I with complete conjugate chromosome pairs and chromosomes distributed along the karyotheca,which were presented as a ring structure by slicing. Only the spreads with 130–150 irregular chromosomes can be easily detected in the 3nDTCC, suggesting that it may undergo an abnormal metaphase I. This study provides new insights into the meiosis of fertile and sterile triploid cyprinid fish.

Further evidence for paternal DNA transmission in gynogenetic grass carp

Gynogenesis is an important breeding method in aquaculture and has been widely applied to many fish species.If gynogenetic progenies are to inherit paternal partial genomic DNA,this will increase genetic variation and will provide a useful outcome for breeding.In this study,we investigated the genetic variation in homeobox(Hox)gene clusters(Hox A4 a,Hox A9 a,Hox A11 b,Hox B1 b,Hox C4 a,Hox C6 b,and Hox D10 a)among koi carp(Cyprinus carpio haematopterus,KOC;the stimulation sperm source),grass carp(Ctenopharyngodon idellus),and gynogenetic grass carp(GGC).We found paternal DNA(a special DNA fragment and Hox C6 b)derived from KOC and a recombinant gene belonging to Hox C6 b in GGC.We are the first to report the recombinant Hox C6 b in GGC.Our study provides further evidence for paternal DNA transmission to gynogenetic progenies,which is a finding with great significance for the genetic breeding of fish.