Aquaculture is becoming the primary source of seafood for human diets, and farmed fish aquaculture is one of its fastest growing sectors. The industry currently faces several challenges including ...infectious and parasitic diseases, reduced viability, fertility reduction, slow growth, escapee fish and environmental pollution. The commercialization of the growth-enhanced AquAdvantage salmon and the CRISPR/Cas9-developed tilapia (
Oreochromis niloticus
) proffers genetic engineering and genome editing tools, e.g. CRISPR/Cas, as potential solutions to these challenges. Future traits being developed in different fish species include disease resistance, sterility, and enhanced growth. Despite these notable advances, off-target effect and non-clarification of trait-related genes among other technical challenges hinder full realization of CRISPR/Cas potentials in fish breeding. In addition, current regulatory and risk assessment frameworks are not fit-for purpose regarding the challenges of CRISPR/Cas notwithstanding that public and regulatory acceptance are key to commercialization of products of the new technology. In this study, we discuss how CRISPR/Cas can be used to overcome some of these limitations focusing on diseases and environmental release in farmed fish aquaculture. We further present technical limitations, regulatory and risk assessment challenges of the use of CRISPR/Cas, and proffer research strategies that will provide much-needed data for regulatory decisions, risk assessments, increased public awareness and sustainable applications of CRISPR/Cas in fish aquaculture with emphasis on Atlantic salmon (
Salmo salar
) breeding.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Sex-specific DNA markers are very helpful for identifying genetic sex and studying sex determination mechanisms in fish. To identify the sex-specific markers of spotted knifejaw (
Oplegnathus ...punctatus
), we performed a comparative analysis of the female and male genomes. In this study, an 18 bp insertion was identified in the male genome after verification by sequencing depth and PCR. An effective and rapid method based on PCR was then developed to identify the genetic sex. A male-female-shared primer pair and a male-specific primer were designed for PCR amplification to avoid false-negative phenomena. To examine the primers in practice, we utilized hundreds of spotted knifejaw fish from different groups to identify their genetic sex, and the results were consistent with their phenotypic sex. The male-specific DNA marker would be helpful for artificial breeding, Y chromosome assembly and further study of the sex determination mechanism. This study is the first to identify an effective sex-specific marker in spotted knifejaw.
Phosphorylation is a post-translational modification that contributes to versatile protein functions in spermatogenesis, and the variations they generate usually results in abnormal spermatogenesis ...or sperm dysfunction. The sex-reversal phenomenon exists in Chinese tongue sole under certain conditions such that individuals with a ZW genotype can acquire a male phenotype and are thus called pseudomales. Pseudomale tongue sole can reach sexual maturity but produce only Z-type sperm, and the Z sperm carries paternal epigenetic information. Whether phosphorylation plays a role in the sperm abnormality of pseudomales is unknown. In this study, a phosphoproteomic analysis was performed to compare protein phosphorylation profiles between pseudomale and male testes. Altogether, we identified 14,253 phosphopeptides matching with 4843 proteins, with 1329 differentially phosphorylated peptides corresponding to 1045 differentially phosphorylated proteins (DPPs). Phosphorylation at 781 sites was upregulated and at 548 sites was downregulated. Four motifs were identified among differentially phosphorylated peptides, which were "SP", "SD", "RxxS", and "TP". Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggested that the cell cycle and DNA/RNA processing were significantly enriched with the genes encoding DPPs. To analyze DPP function in depth, a protein-protein interaction network was constructed, and Ran-binding protein 2 was found to play a central role in spermatogenesis by regulating several processes such as the cell cycle, eukaryotic translation, ubiquitination, and minichromosome maintenance. In kinase-associated network analyses, two "mitogen-activated protein kinase (Mapk)-centered" clusters were identified that may account for abnormal spermatogenesis in pseudomales. One cluster was centered on Mapk6, which predominantly regulated the cell cycle by interacting with several cyclin-dependent kinases, and the other was centered on the "testis-expressed kinase 1-like (Tesk1l)/Pim1l-Mapk4l- testis-expressed 14 (Tex14)" kinase cascade, which might contribute to spermatogenesis by regulating β-catenin. Taken together, these data suggested the new candidates involved in pseudomale sperm abnormalities and provided clues to discover the phosphorylated regulatory mechanism underlying tongue sole spermatogenesis.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The thylakoid membrane system of higher plant chloroplasts consists of interconnected subdomains of appressed and nonappressed membrane bilayers, known as grana and stroma lamellae, respectively. ...CURVATURE THYLAKOID1 (CURT1) protein complexes mediate the shape of grana stacks in a dosage-dependent manner and facilitate membrane curvature at the grana margins, the interface between grana and stroma lamellae. Although grana stacks are highly conserved among land plants, the functional relevance of grana stacking remains unclear. Here, we show that inhibiting CURT1-mediated alteration of thylakoid ultrastructure in Arabidopsis (Arabidopsis thaliana) reduces photosynthetic efficiency and plant fitness under adverse, controlled, and natural light conditions. Plants that lack CURT1 show less adjustment of grana diameter, which compromises regulatory mechanisms like the photosystem II repair cycle and state transitions. Interestingly, CURT1A suffices to induce thylakoid membrane curvature in planta and thylakoid hyperbending in plants overexpressing CURT1A. We suggest that CURT1 oligomerization is regulated at the posttranslational level in a light-dependent fashion and that CURT1-mediated thylakoid plasticity plays an important role in fine-tuning photosynthesis and plant fitness during challenging growth conditions.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The complement system is important in the innate immune response. C1q-domain-containing proteins have multiple functions and occur extensively in invertebrates and vertebrates. In this study, PoC1ql3 ...encoding a C1q-domain-containing protein in the Japanese flounder was identified. The 266-amino-acid polypeptide encoded, PoC1ql3, shares high sequence and structural similarity with orthologues in other fish and mammals. PoC1ql3 is abundantly expressed in the brain, but less in the blood, gills, and liver. Transcripts of PoC1ql3 were down-regulated in the spleen and liver 6–24 h after bacterial infection, but were significantly up-regulated after 48 h. Full-length PoC1ql3 (C1ql3-full) and its gC1q domain (C1ql3-part) were both exerted anti-Edwardsiella tarda activity. C1ql3-part bound to lipopolysaccharide and peptidoglycan, and exerted antibacterial effects against E. tarda in vivo, suggesting that C1ql3 functions as a pathogen-recognition receptor. Therefore, PoC1ql3 functions in the innate immune system, which would facilitate the investigation of the immune system in Japanese flounder.
•Characterization of a novel C1q-domain-containing protein of the Japanese flounder.•PoC1ql3 was expressed not only in the brain, but also in many immune-related tissues.•C1ql3-full and C1ql3-part protein exerted antimicrobial function against E. tarda in vitro.•The C1ql3-part bound LPS and PGN as pattern-recognition receptor.•The C1ql3-part inhibited the invasion of bacterium to fish kidney cells in vivo.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The Chinese tongue sole (
) is a traditional, precious fish in China. Due to the large growth difference between males and females, the investigation of their sex determination and differentiation ...mechanisms receives a great deal of attention. Forkhead Box O (
) plays versatile roles in the regulation of sex differentiation and reproduction. Our recent transcriptomic analysis has shown that
genes may participate in the male differentiation and spermatogenesis of Chinese tongue sole. In this study, six
members (
,
,
,
,
, and
) were identified. Phylogenetic analysis indicated that these six members were clustered into four groups corresponding to their denomination. The expression patterns of the gonads at different developmental stages were further analyzed. All members showed high levels of expression in the early stages (before 6 months post-hatching), and this expression was male-biased. In addition, promoter analysis found that the addition of C/EBPα and c-Jun transcription factors enhanced the transcriptional activities of
,
,
, and
The siRNA-mediated knockdown of the
,
, and
genes in the testicular cell line of Chinese tongue sole affected the expression of genes related to sex differentiation and spermatogenesis. These results have broadened the understanding of
's function and provide valuable data for studying the male differentiation of tongue sole.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The sex chromosome, especially specific in one sex, generally determines sexual size dimorphism (SSD), a phenomenon with dimorphic sexual difference in the body size. For
, a flatfish in China, ...although the importance of chromosome W and its specific gene
in female-biased SSD have been suggested, its family members and regulation information are still unknown. At present, three
copies gene were identified on chromosome W, with no gametologs. Phylogenetic analysis for the ZBED family revealed an existence of ZBED9 in the fish. Nine members were uncovered from
, clustering into three kinds, ZBED1, ZBED4 and ZBEDX, which is less than the eleven kinds of ZBED members in mammals. The predominant expression of
in the female brain and pituitary tissues was further verified by qPCR. Transcription factor
could significantly enhance the transcriptional activity of
promoter, which is opposite to its effect on the male determinant factor-
. When
was interfered with,
,
and
were up-regulated, while cell-cycle-related genes, including
and
were down-regulated. Thus,
is involved in cell proliferation by regulating
,
, cell cycle and the Wnt pathway. Further research on their interactions would be helpful to understand fish SSD.
The high proportion of males in
hinders their industrial development. The genetic ZW individual can become a pseudomale by sex reversal. And the pseudomale can produce Z-sperm (with epigenetic ...information to cause sex reversal) while W-sperm is absent, which leads to an even higher male proportion in offspring. Recently, with the development of transcriptomic technologies, research on spermatogenesis in
has been focused on the ubiquitination pathway. In this study, we analyzed the function of the ubiquitin ligase
gene on the Z chromosome. A qPCR experiment showed that its expression level in the gonad was the highest among different tissues. In the ovary, the expression gradually increased with development from 40 days post-hatching (dph) to 1.5 years post-hatching (yph). In the testis,
showed increased expression from 40 dph to 6 months post-hatching (mpf) and stabilized up until 1.5 ypf. In situ hybridization showed that the mRNA of
was mainly distributed in the germ cells of the testis and the ovary. In vivo siRNA-mediated knockdown of the
gene in male fish affected the expression of a series of genes related to sex differentiation and spermatogenesis. These results provide genetic data on the molecular mechanisms of gonadal development and spermatogenesis in
.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Abstract
Turbot (
Scophthalmus maximus
), commercially important flatfish species, is widely cultivated in Europe and China. With the continuous expansion of the intensive breeding scale, turbot is ...exposed to various stresses, which greatly impedes the healthy development of turbot industry. Here, we present an improved high-quality chromosome-scale genome assembly of turbot using a combination of PacBio long-read and Illumina short-read sequencing technologies. The genome assembly spans 538.22 Mb comprising 27 contigs with a contig N50 size of 25.76 Mb. Annotation of the genome assembly identified 104.45 Mb repetitive sequences, 22,442 protein-coding genes and 3,345 ncRNAs. Moreover, a total of 345 stress responsive candidate genes were identified by gene co-expression network analysis based on 14 published stress-related RNA-seq datasets consisting of 165 samples. Significantly improved genome assembly and stress-related candidate gene pool will provide valuable resources for further research on turbot functional genome and stress response mechanism, as well as theoretical support for the development of molecular breeding technology for resistant turbot varieties.
Phosphatidylinositol transfer protein (pitp) plays an important role in phospholipid transfer in animals. A pitp variant (
) in Chinese tongue sole was identified by transcriptomic analysis for its ...female-biased expression. The coding sequence of
was 816 bp, encoding a 371-amino-acid protein.
showed female-biased expression and was relatively high in brain, muscle, and ovary tissues. In different developmental stages of the ovary,
could be detected from 40 days until 3 years post hatching, and the highest expression was observed at 90 days.
hybridization revealed that
was predominantly localized in early-stage oocytes (I-III stages). After siRNA-mediated knockdown of
in an ovarian cell line, the expression of
was reduced, while that of
and
was significantly increased. Our findings suggest that
might be involved in female differentiation and early oogenesis.
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