Marine Biotechnology最新文献

Specific cell depletion is a common means to study the physiological function of cell lineages and tissue regeneration. However, 100% depletion is difficult to achieve with existing cell depletion strategies. With the increasing maturity of CRISPR/Cas9 technology, it is increasingly used for the depletion of various cells. However, even with this technology, it is difficult to complete the depletion of specific gene knockout cells. For this reason, cell depletion with the use of repetitive sequences as the target of CRISPR/Cas9 was explored using zebrafish. All cells were used as the target cells for the first set of experiments. The results showed that injection of a mixture of DANA-gRNA and Cas9 mRNA into zygotes resulted in substantial cell apoptosis. Cells are almost invisible in the embryonic animal pole during the dome stage. The activities of the caspase-3 and caspase-9 proteins and the mRNA level of the P53 gene were significantly increased. Then, primordial germ cells (PGCs) in embryos were used as the target cells in subsequent experiments. To specifically knock out PGCs, we injected the mix of DANA-gRNA, pkop: Cas9 plasmid (the kop promotor allows Cas9 expression only in PGCs), and eGFP-nos3′UTR mRNA into zebrafish fertilized eggs. The results revealed that the activity of the caspase-3 protein was significantly increased, and the mRNA levels of P53, ku70, and ku80 were significantly upregulated, while the number of PGCs decreased gradually. Few PGCs labeled with GFP could be seen 20 h post-fertilization (hpf), and no PGCs could be seen at the germinal ridge 24 hpf. Therefore, the combination of CRISPR/Cas9 technology and repetitive sequences can achieve efficient cell depletion regardless of whether there is generalized expression or expression in specific cells. These results indicate that it is feasible to eliminate cells by using repeat sequences as CRISPR/Cas9 system target sites.

The molluscan family Ostreidae, commonly known as oysters, is an important molluscan group due to its economic and ecological importance. In recent years, an abundance of genomic data of Ostreidae species has been generated and available in public domain. However, there is still a lack of a high-efficiency database platform to store and distribute these data with comprehensive tools. In this study, we developed an oyster genome database (OysterDB) to consolidate oyster genomic data. This database includes eight oyster genomes and 208,923 protein-coding gene annotations. Bioinformatic tools, such as BLAST and JBrowse, are integrated into the database to provide a user-friendly platform for homologous sequence searching, visualization of genomes, and screen for candidate gene information. Moreover, OysterDB will be continuously updated with ever-growing oyster genomic resources and facilitate future studies for comparative and functional genomic analysis of oysters (http://oysterdb.com.cn/).

The aim of this study was to investigate the effects of melatonin (MT) feed supplementation on the antioxidant capacity, immune defense, and intestinal flora in Procambarus clarkii (P. clarkii). Six groups of P. clarkii were fed test feeds containing different levels of MT: 0 mg/kg (control), 22.5, 41.2, 82.7, 165.1, and 329.2 mg/kg for a duration of 2 months. The specific growth rate, hepatosomatic index, and condition factor were recorded highest in the test group of shrimp fed an MT concentration of 165.1 mg/kg. Compared to the control group, the rate of apoptosis was lower in hepatopancreas cells of P. clarkii supplemented with high concentrations of MT. Analyses of antioxidant capacity and immune-response-related enzymes in the hepatopancreas indicated that dietary supplementation of MT significantly augmented both the antioxidant system and immune responses. Dietary MT supplementation significantly increased the expression levels of antioxidant-immunity-related genes and decreased the expression levels of genes linked to apoptosis. Dietary MT was associated with an elevation in the abundance of the Firmicutes and a reduction in the abundance of the Proteobacteria in the intestines; besides, resulting in an increase in the abundance of beneficial bacteria, such as Lactobacilli. The broken-line model indicated that the suitable MT concentration was 154.09–157.09 mg/kg. MT supplementation enhanced the growth performance of P. clarkii, exerting a positive influence on the intestinal microbiota, and bolstered both immune response and disease resistance. Thus, this study offered novel perspectives regarding the application of dietary MT supplementation within the aquaculture field.

Bicarbonate and sulfate are among two primary ion constituents of saline-alkaline water, with excessive levels potentially causing metabolic disorders in crustaceans, affecting their molting and interrupting development. As an economically important crustacean species, the molecular adaptive mechanism of giant freshwater prawn Macrobrachium rosenbergii in response to the stress of bicarbonate and sulfate remains unexplored. To investigate the mechanism underlying NaHCO₃, Na₂SO₄, and mixed NaHCO₃, Na₂SO₄ stresses, M. rosenbergii larvae were exposed to the above three stress conditions, followed by total RNA extraction and high-throughput sequencing at eight distinct time points (0, 4, 8, 12, 24, 48, 72, and 96 h). Subsequent analysis revealed 13, 16, and 13 consistently identified differentially expressed genes (DEGs) across eight time points under three stress conditions. These consistently identified DEGs were significantly involved in the Gene Ontology (GO) terms of chitin-based cuticle development, protein-carbohydrate complex, structural constituent of cuticle, carnitine biosynthetic process, extracellular matrix, and polysaccharide catabolic process, indicating that alkaline stresses might potentially impact the energy metabolism, growth, and molting of M. rosenbergii larvae. Particularly, the transcriptome data revealed that DEGs associated with energy metabolism, immunity, and amino acid metabolism were enriched across multiple time points under three stress conditions. These DEGs are linked to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including glycolysis/glucogenesis, amino sugar and nucleotide sugar metabolism, and lysine degradation. Consistent enrichment findings across the three stress conditions support conclusions above. Together, these insights are instrumental in enhancing our understanding of the molecular mechanisms underlying the alkaline response in M. rosenbergii larvae. Additionally, they offer valuable perspectives on the regulatory mechanisms of freshwater crustaceans amid saline-alkaline water development.

To assess the impact of different substrates in a recirculating water system on the immune response and antioxidant capacity of Babylonia areolata, we conducted a comparative analysis of the transcriptomes and antioxidant performance of the digestive glands in three substrate environments (sand—S group, ceramic granules—C group, and PVC breeding nest—P group). Transcriptome results revealed that the S group and P group exhibited the highest number of differentially expressed genes (DEGs), with a total of 2218 DEGs, including 928 upregulated and 1290 downregulated DEGs. The C group and P group had 1055 DEGs in common, with 316 upregulated and 739 downregulated DEGs. The C group and S group had the fewest DEGs, with 521 in total, including 303 upregulated and 218 downregulated DEGs. GO enrichment analysis showed that in the S vs P group, terms such as catalytic activity, membrane part, and cellular process were enriched with 287, 262, and 180 DEGs, respectively. In the C vs P group, binding, cellular process, and cell part were enriched with 146, 135, and 127 DEGs, respectively. In the C vs S group, catalytic activity, membrane part, and metabolic process were enriched with 90, 83, and 59 DEGs, respectively. Kegg enrichment analysis revealed significant changes in immune-related pathways in the S vs P group, including lysosome, phagosome, and leukocyte transendothelial migration, with 30, 13, and 10 enriched DEGs, respectively. In the C vs P group, phagosome, drug metabolism—other enzymes, and N-Glycan biosynthesis showed significant changes in immune-related pathways, with 9, 6, and 4 enriched DEGs, respectively. In the C vs S group, lysosome, PPAR signaling pathway, and fatty acid degradation exhibited significant changes in immune-related pathways, with 8, 4, and 3 enriched DEGs, respectively. Regarding antioxidant capacity, the S group showed significantly higher total T-AOC than the other experimental groups, while CAT, SOD, POD, and AKP were lower than in the C and P groups. The ACP level in the Sand group was not significantly different from the P group but significantly lower than the C group. In conclusion, substrate environments significantly influence the immune-related genes and key antioxidant enzyme activities in B. areolata.

Environmental pollution is a significant problem due to the improper disposal of plastics and shrimp shells outdoors. Therefore, the synthesis of biodegradable film from waste materials is highly important. The novelty of this research lies in the extraction of protein hydrolysates and chitosan from shrimp shells, as well as the fabrication of biodegradable film from these materials. In this study, the composite films were produced using the solution casting method. Moreover, the combined effect of ultrasound pretreatments (UPT) and natural deep eutectic solvents (NADES) was investigated as extraction media, to determine their potential impact on shrimp waste subcritical water hydrolysis (SWH). Shrimp shells were submitted to UPT in NADES solution, followed by SWH at different temperatures ranging from 150 to 230 °C under 3 MPa for 20 min. Then, the physiochemical properties and bioactivities of the hydrolysates were assessed to determine their suitability for use in biodegradable packaging films. Additionally, the physiochemical properties and bioactivities of the resulting hydrolysates were also analyzed. The highest amount of protein (391.96 ± 0.48 mg BSA/g) was obtained at 190 °C/UPT/NADES, and the average molecular size of the protein molecules was less than 1000 Da with different kinds of peptide. Overall, combined UPT and SWH treatments yielded higher antioxidant activity levels than individual treatments. Finally, the application of composite films was evaluated by wrapping fish samples and assessing their lipid oxidation. The use of higher concentrations of protein hydrolysates significantly delayed changes in the samples, thereby demonstrating the film’s applicability.

The use of sterile recipients is crucial for efficiently producing donor-derived offspring through surrogate broodstock technology for practical aquaculture applications. Although knockout (KO) of the dead end (dnd) gene has been used in previous studies as a sterilization method, it has not been reported in marine fish. In this study, nibe croaker was utilized as a model for marine teleosts that produce small pelagic eggs, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized to produce dnd KO fish. The F1 generation, which carried a nonsense mutation in the dnd gene, was produced by mating founder individuals with wild-type counterparts. Subsequently, the F2 generation was produced by mating the resulting males and females. Among the F2 generations, 24.0% consisted of homozygous KO individuals. Histological analysis revealed that primordial germ cells (PGCs) were present in homozygous KO individuals at 10 days post-hatching (dph), similar to wild-type individuals. However, by 20 dph, PGCs were absent in KO individuals. Furthermore, no germ cells were observed in the gonads of both sexes of homozygous KO individuals at 6 months old, which is the typical maturity age for wild-type individuals of both sexes. In addition, when cryopreserved donor nibe croaker testicular cells were transplanted, only donor-derived offspring were successfully obtained through the spontaneous mating of homozygous KO recipient parents. Results indicate that dnd KO nibe croaker lacks germ cells and can serve as promising recipients, producing only donor-derived gametes as surrogate broodstock.

Marine environments harbor a plethora of microorganisms that represent a valuable source of new biomolecules of biotechnological interest. In particular, enzymes from marine bacteria exhibit unique properties due to their high catalytic activity under various stressful and fluctuating conditions, such as temperature, pH, and salinity, fluctuations which are common during several industrial processes. In this study, we report a new esterase (EstGoM) from a marine Pseudomonas sp. isolated at a depth of 1000 m in the Gulf of Mexico. Bioinformatic analyses revealed that EstGoM is an autotransporter esterase (type Va) and belongs to the lipolytic family II, forming a new subgroup. The purified recombinant EstGoM, with a molecular mass of 67.4 kDa, showed the highest hydrolytic activity with p-nitrophenyl octanoate (p-NP C8), although it was also active against p-NP C4, C5, C10, and C12. The optimum pH and temperature for EstGoM were 9 and 60 °C, respectively, but it retained more than 50% of its activity over the pH range of 7–11 and temperature range of 10–75 °C. In addition, EstGoM was tolerant of up to 1 M NaCl and resistant to the presence of several metal ions, detergents, and chemical reagents, such as EDTA and β-mercaptoethanol. The enzymatic properties of EstGoM make it a potential candidate for several industrial applications.

Myostatin (MSTN, also known as growth differentiation factor-8 (GDF-8)), a member of the transforming growth factor β (TGF-β) superfamily, functions as a negative regulator of skeletal muscle development and growth. However, it is also expressed in a wide range of tissues in fish and thus may have more diverse roles in this group than in mammals. In this study, we assessed the genome-wide transcriptional expression pattern associated with the CRISPR/Cas9-mutated MSTN gene in the olive flounder (Paralichthys olivaceus) in association with changes in cell proliferation and transportation processes. There were no differences in the hepatosomatic index, and the growth of male and female fish increased in the F1 progeny of the MSTN mutants. Furthermore, the histopathological analysis showed that myostatin editing resulted in a 41.24% increase in back muscle growth and 46.92% increase in belly muscle growth in male flounder compared with normal flounder, and a 16.01% increase in back muscle growth and 14.26% increase in belly muscle growth in female flounder compared with normal flounder. This study demonstrates that editing of the myostatin gene enhances muscle growth in olive flounder, with a notably more pronounced effect observed in males. Consequently, myostatin-edited male flounder could represent a valuable asset for the flounder aquaculture industry.

The potential functional role(s) of heat shock protein 70 (Hsp70) in the brine shrimp, Artemia franciscana, a crucial crustacean species for aquaculture and stress response studies, was investigated in this study. Though we have previously reported that Hsp70 knockdown may have little or no impact on Artemia development, the gestational survival and number of offspring released by adult females were impaired by obscuring Hsp70 synthesis. Transcriptomic analysis revealed that several cuticle and chitin synthetic genes were downregulated, and carbohydrate metabolic genes were differentially expressed in Hsp70-knockdown individuals. A more comprehensive microscopic examination performed in this study revealed exoskeleton structural destruction and abnormal eye lenses featured in Hsp70-deficient adult females 48 h after Hsp70 dsRNA injection. Cysts produced by these Hsp70-deficient broods, instead, had a defective shell and were smaller in size, whereas nauplii had shorter first antennae and a rougher body epicuticle surface. Changes in carbohydrate metabolism caused by Hsp70 knockdown affected glycogen levels in adult Artemia females, as well as trehalose in cysts released from these broods, indicating that Hsp70 may play a role in energy storage preservation. Outcomes from this work provided novel insights into the roles of Hsp70 in Artemia reproduction performance, cyst formation, and exoskeleton structure preservation. The findings also support our previous observation that Hsp70 knockdown reduced Artemia nauplius tolerance to bacterial pathogens, which could be explained by the fact that loss of Hsp70 downregulated several Toll receptor genes (NT1 and Spaetzle) and reduced the integrity of the exoskeleton, allowing pathogens to enter and cause infection, ultimately resulting in mortality.