Marine Biotechnology最新文献

Induced pluripotent stem cells (iPSCs) are a new type of pluripotent cells reprogrammed from somatic cells back into an embryonic-like pluripotent state of stem cells to study development, disease and potential gene therapies. The induction and regulation mechanisms of iPSCs in fish are still unclear. By using the transfection technique, we investigated the crucial function of the OSKMNL factor co-expression for somatic reprogramming in the muscle cell line of large yellow croaker (Larimichthys crocea) (LYCMs) and successfully established a stable iPSCs line (Lc-OSNL-iPSCs). Stable culturing of iPSCs with high alkaline phosphatase activity and a stable karyotype was achieved. The qRT-PCR and immunofluorescence labeling results revealed that Lc-OSNL-iPSCs displayed a high expression level of pluripotent marker genes such as Nanog, Oct4, and Sox2. There were significant differences between Lc-OSNL-iPSCs, Lc-OSKMNL-iPSCs, and LYCMs, and the expression of several genes in maintaining cell pluripotency was up-regulated when the pluripotency signal pathway of stem cells was activated. The technical system for inducing iPSCs of Larimichthys crocea was constructed in this study. This system can serve as a basic model to understand germ cell differentiation mechanism, gender control, genetics, and breeding of large yellow croaker and a platform for studying iPSCs in fish. Interestingly, the acquired iPSCs serves as a useful material for the directional induction of muscle stem cells, thereby establishing the groundwork for obtaining "artificial fish" in the future.

Graphical Abstract

Cyclin-dependent kinases (Cdks) are major molecules related to cell cycle regulation. Polyploidy can be caused by the production of unreduced gametes, which is often related to the abnormal cell cycle of germ cells. Here, we successfully constructed a cdk1 mutation line (cdk1^+/−) in zebrafish, a commonly used model organism. It showed that cdk1 depletion resulted in the generation of both polyploid and aneuploid embryos of WT♀ × cdk1^+/−♂ zebrafish. In addition to normal sperms (1N), the depletion of cdk1 in zebrafish also led to the production of some large-head 2N sperms and higher ploidy sperms. Results of bivalent analysis of testis and ultrastructure analysis of spermatogonia suggested that the production of these large-head sperms was due to spermatogonia chromosome doubling in cdk1^+/− zebrafish. Transcriptome analysis revealed aberrant expressions of some cell cycle and DNA replication-related genes in the early testis of cdk1^+/− zebrafish relative to WT zebrafish. Through STRING correlation analysis, we further proved that cdk1 depletion affected the mitosis process and endoduplication initiation of spermatogonia by regulating expressions of some proteins related to cell cycle (i.e., Espl1 and Pp1) and DNA replication (i.e., Orc1 and Rnaseh2b), thereby leading to the formation of unreduced sperms. This study provides important information on revealing the molecular mechanisms of unreduced gamete formation caused by cdk1 mutation. Meanwhile, it also provides an important reference for the creation of fish polyploid germplasm.

The severity, frequency, and duration of extreme events, in the context of global warming, have placed many marine ecosystems at high risk. Therefore, the application of methods that can mediate the impacts of global warming on marine organisms seems to be an emerging necessity in the near term. In this context, enhancing the thermal resilience of marine organisms may be crucial for their sustainability. It has been shown that the repeated time-limited exposure of an organism to an environmental stimulus modifies its response mode, thus enhancing resilience and allowing adaptation of the physiological and developmental phenotype to environmental stress. In the present study, we investigated the “stress memory” effect caused by heat hardening on Mytilus galloprovincialis cellular pathways to identify the underlying biochemical mechanisms that enhance mussel thermal tolerance. Heat hardening resulted in increased ETS activity and ATP production and increased autophagic performance at all elevated temperatures (24 °C, 26 °C, and 28 °C). Furthermore, at these increased temperatures, apoptosis and inflammation remain at significantly lower levels in pregnant individuals than in nonhardened individuals. Autophagy, as a negative regulator of apoptosis, may lead to decreased damage to surrounding cells, which in turn alleviates inflammatory effects. In conclusion, the exposure of mussels to heat hardening seems to provide a physiological response that enhances heat tolerance and increases cell survival through increased energy production and reduced cell death and inflammatory responses. The latter can be utilized for the management and conservation of aquatic species of economic value or endangered status.

As human activities increase and environmental changes persist, increased ultraviolet B (UVB) radiation in aquatic ecosystems poses significant threats to aquatic life. This study, through transcriptomic analysis of the mantle tissue of Crassostrea gigas following UVB radiation exposure, identified and validated two key transcription factors, CgRunx1 and CgCBFβ. The highest expression levels of CgRunx1 and CgCBFβ in the mantle suggest their pivotal roles in this tissue. Co-immunoprecipitation experiments revealed that CgRunx1 and CgCBFβ could form heterodimers and interact with each other. Furthermore, this study assessed the impact of UVB radiation on the levels of reactive oxygen species of the C. gigas, speculating that CgRunx1, as a potential redox-sensitive transcription factor, might be regulated by intracellular ROS. Through screening and binding site prediction analysis of target genes, coupled with dual-luciferase reporter assays, we verified that CgRunx1 might participate in regulating the biomineralization and autophagy processes in C. gigas by activating the transcriptional expression of target genes Transport and Golgi organization 1 and V-type proton ATPase catalytic subunit A. These findings provide new insights into the molecular response mechanisms of the C. gigas to UVB radiation and lay an important foundation for studying the adaptive evolution of bivalves to environmental stress.

Apostichopus japonicus is an important marine aquaculture species in China, with high nutritional and economic value. In A. japonicus, there is no obvious sexual dimorphism in external appearance, and sex differentiation primarily relies on the observation of mature gonads after dissection, which leads to difficulties in sex identification. The confusion in sex identification greatly reduces breeding efficiency in the sea cucumber industry. Therefore, developing a reliable sex-specific marker is crucial. In this study, we identified 586 male-specific sequences through whole-genome assembly and sequence alignment, but did not identify any female-specific sequences, inferring an XY-type sex determination system in sea cucumbers. We developed a set of male-specific molecular markers to establish an accurate, stable, and widely adaptable genetic sex identification technique for A. japonicus. The male-specific molecular markers were validated with 100% accuracy in sea cucumber populations from six different geographical regions in China. In conclusion, this study provides further evidence for the XY-type sex determination system in A. japonicus and establishes an effective genetic sex identification method for multi-geographic populations, which benefits future study on reproductive biology and has significant implications in sea cucumber aquaculture industry.

This study facilitates design of expression vectors and lentivirus tools for gene editing of Atlantic salmon. We have characterized widely used heterologous promoters and novel endogenous promoters in Atlantic salmon cells. We used qPCR to evaluate the activity of several U6 promoters for sgRNA expression, including human U6 (hU6), tilapia U6 (tU6), mouse U6 (mU6), zebrafish U6 (zU6), Atlantic salmon U6 (sU6), medaka U6 (medU6), and fugu U6 (fU6) promoters. We also evaluated several polymerase type II (pol II) promoters by luciferase assay. Our results showed that hU6 and tU6 promoters were the most active among all the tested U6 promoters, and heterologous promoters (CMV, hEF1α core) had higher activity compared to endogenous Atlantic salmon promoters sHSP8, sNUC3L, sEF1α. Among endogenous pol II promoters, sEF1α and sHSP8 displayed higher activity than sNUC3L, sHSP703, sHSP7C, sXRCC1L, and sETF. We observed that extending the promoter sequence to include the region up to the start codon (ATG) resulted in a significant increase in expression efficiency for sNUC3L and sEF1α. We also show that mutating the PRDM1 motif will significantly decrease the activity of the sEF1α promoter. The presence of the PRDM1 motif in sHSP8 promoter was also associated with relatively high expression compared to the promoters that naturally lacked this motif, such as sNUC3L. We speculate that this short sequence might be included in other promoters to further enhance the promoter activity, but further experiments are needed to confirm this. Our findings provide valuable insights into the activity of different promoters in Atlantic salmon cells and can be used to facilitate further transgenic studies and improve the efficiency of transgene expression in Atlantic salmon.

Spotted knifejaw (Oplegnathus punctatus), one of the most valuable mariculture species, grows with significant sexual dimorphism, with males growing significantly faster than females. O. punctatus not only has excellent growth characteristics and high food value, but also shows high economic value in aquaculture, which has become a hotspot in the field of aquaculture. The current insufficiency of sex marker identification in O. punctatus restricts the process of its unisexual breeding. Rapid identification of sex will help to study the mechanisms of sex determination and accelerate the development of sex-controlled breeding. With the completion of the sequencing of the male and female genomes of O. punctatus, the efficient and precise development of genetic sex markers has been made possible. In this study, we used genome-wide information combined with molecular biology techniques from marker sequences to further establish a rapid method for DNA insertion variant detection in the intron of O. punctatus erc2 gene, which can be used to rapidly, accurately, and efficiently identify whether DNA insertion occurs in the intron of O. punctatus erc2 gene to be detected, and to identify the sex of O. punctatus to be detected. It could also be distinguished by agarose gel electrophoresis, which would shorten the time for accurate identification and improves the detection efficiency. Homozygous comparison of male and female individuals showed that the length of the DNA fragment of the erc2 gene was 239 bp on chromosome X₁ and 1173 bp on chromosome Y. It can therefore be inferred that a 934 bp insertion fragment exists on the Y chromosome. The PCR amplification results showed that two DNA fragments of 1173 bp and 239 bp could be amplified in male O. punctatus, and the 1173 bp fragment was a marker fragment specific to the variant intron erc2 gene, while only a single DNA fragment of 239 bp was amplified in female O. punctatus. It has important significance and application value in the study of neurotransmitter transmission and environmental adaptability of female and male fish based on erc2 gene, as well as the identification of male and female sex, the preparation of high male fry, and family breeding.

In this study, we examined the effect of Laminaria japonica polysaccharide (fucoidan) on the regulation of lipid metabolism. A rat model of diabetes mellitus (DM) was established by a high-sugar and high-fat diet combined with streptozotocin. Changes in the rats’ body weight and blood glucose level during the experiment were recorded. Before the end of the experiment, an automatic biochemical analyzer was used to detect the fasting blood glucose (FBG), lipid content in serum, and insulin content, and calculate the insulin resistance index. Oil red O staining was used to detect lipid deposition in the liver. H&E staining, Masson staining, and PASM staining were used to observe the pathological structural changes in the liver. 16 s RNA sequencing and targeted metabolomics were used to detect intestinal microbiota and bile acid content. The results showed that fucoidan was able to inhibit weight loss in the DM rats and reduce the content of triglycerides (TG), cholesterol (TC), and low-density lipoprotein (LDL-C) in serum. Oil red O staining showed a decrease in liver fat accumulation after fucoidan treatment. 16 s RNA sequencing demonstrated that fucoidan increased the abundance of Bacteroidia, Campylobacteria, Clostridia, Gammaproteobacteria, Negativicutes, and Verrucomicrobi. Fucoidan also increased the secretion of secondary bile acids (Nor-DCA, TLCA, β-UDCA) and alleviated lipid metabolism disorders. The expression of α-SMA was inhibited by fucoidan, whereas the expression of FXR and TGR5 was promoted. Fucoidan shows good activity in regulating lipid metabolism by regulating the expression of FXR and TGR5 and acting on the intestinal flora–bile acid axis.

Heat stress is a major problem in aquaculture species, causing changes in physiology such as decreased feed intake, growth rate, reproduction, and internal cellular damage, thereby affecting fish’s health. The effects of an acute heat stress simulating a daily rise and fall in temperature on summer days were evaluated in the liver proteome of rohu (Labeo rohita) fingerlings in the present study. The fish maintained at 30 °C were gradually exposed to a higher temperature of 36 °C at an increment rate of 1 °C per 1.5 h, and after 3 h at that temperature, it was gradually reduced to 30 °C. The liver tissue samples were collected at 5 am, 5 pm, and 5 am the next day from the exposed and control fish. Protein samples were prepared from the liver tissues, and the extracted proteins were compared using 2-dimensional (2D) gel electrophoresis (2DGE) and mass spectrometry (MS) using a MALDI-TOF/TOF mass spectrometer. A total of 44 differentially expressed protein spots were visualized in 2D gel analysis from heat stress exposed fish at three time points, out of which 21 proteins including one hypothetical protein could be identified by MS. The abundance of five selected differentially expressed proteins (DEPs) was validated using qPCR. The majority of DEPs were found to be involved primarily in lipid, protein and energy metabolism, immune system regulation, cytoskeletal stability, and ROS management. The findings of this study would help in the development of strategies to mitigate heat stress in L. rohita.

The common clownfish, Amphiprion ocellaris, is an iconic coral reef fish, ubiquitous in the marine aquarium hobby and useful for studying a variety of biological processes (e.g., mutual symbiosis, ultraviolet vision, and protandrous sex change). Recently, CRISPR/Cas9 methods were developed for knocking out specific genes for mechanistic studies. Here, we expand the genetic toolkit for A. ocellaris by creating the first transgenic line using the Tol2 transposon system. Fertilized eggs were co-injected with Tol2 transposase mRNA and a plasmid encoding an elongation factor-1α (Ef1α): green fluorescent protein (GFP) cassette at various concentrations, needle tip dimensions, and timepoints post-fertilization. We compared various injection parameters and sterilization methods to maximize the survival of injected eggs. F0s (n = 10) that were genotyped GFP + were then raised to 6 months of age and crossed with wild-type (WT) females to confirm germline transmission. F1 offspring were also raised and crossed in the same manner. The highly efficient Tol2 transposon system resulted in a 37% rate of transgenesis for surviving eggs amounting to a 2.7% yield of all injected eggs surviving and being GFP + (n = 160). Of these, 10 were raised to adulthood, 8 spawned, and 5/8 (62.5%) produced GFP + offspring. Further, two F1s crossed with WT females produced 54.2% and 44.6% GFP + offspring respectively, confirming the creation of a stable line. This is, to our knowledge, the first generation of a transgenic line in any coral reef fish. The ability to express transgenes of interest in the iconic anemonefish opens the door to a new era of exploration into their fascinating biology.