Marine Biotechnology最新文献

Abalone, a marine mollusk with significant economic and ecological value, plays a crucial role in sustainable aquaculture. The development and application of CRISPR-Cas9 gene-editing technology have opened up a new path for improving breeding efficiency. CRISPR/Cas9-mediated gene editing has been achieved in abalones via microinjection. In this study, a gene encoding myostatin MSTN in H. discus hannai; was selected as target for conducting the CRISPR-Cas9 gene editing experiment in combination with an electroporation delivery system. Our results showed that all three sgRNAs effectively targeted and cleaved the target segment, with sgRNA1 and sgRNA2 exhibiting high in vitro activity. After electroporation, the effects of transfection on embryonic development of fertilized eggs were observed and statistically analyzed. 12.7 ± 5.4% of the fertilized eggs were damaged and deformed after electroporation. Twenty-four hours after electroporation, surviving larvae were collected for DNA extraction and sequencing. Two potential mutations within the target region of MSTN were identified by sequencing. These results provide a reference for the improvement and development of CRISPR-mediated gene editing methods in marine mollusks such as abalones.

Tetrodotoxin (TTX), also known as puffer fish venom, has gained wide popularity in recent years as an effective tool in the field of physiology, as well as a promising anesthetic and analgesic agent. However, to date, no economically viable method of TTX production has been found. In this work, using high-performance liquid chromatography with tandem mass spectrometry, we conducted a study of the dynamics of the content and ratio of TTX and its analogues (TTXs) in the eggs of captive nemerteans of the Cephalothrix simula species complex, as well as a study of TTX localization in nemertean eggs using confocal laser scanning microscopy. It has been shown that nemertean eggs and nemerteans themselves, when kept in captivity for a long time, can contain concentrations of TTXs comparable to individuals of the wild population. It was found that TTX in eggs is associated with yolk granules, for which morphological characterization was carried out for the first time. The data obtained in the work can be used as a basis for the development of a technique for keeping animals in captivity for as long as possible while maintaining/increasing their toxic potential for further toxin extraction from egg yolk.

Channa striata is a hardy fish that can thrive in various aquatic conditions; it is a good source of protein and has high economic value. This study was conducted to establish and characterize the muscle cell line of C. striata and use it to develop micro-tissue using seaweed biofilm. Explant culture was used to create a continuous muscle cell line from C. striata, which was then sub-cultured 143 times in Leibovitz L-15 medium with 10% FBS at 28 °C. They were properly cryopreserved, and after storage, 88–92% of the cells were recovered. The C. striata muscle cell line (CSM) was authenticated by examining the mitochondrial 16S rRNA gene using polymerase chain reaction. The immunophenotyping results revealed that the CSM cells were of myoblast origin, as evidenced by desmin and myosin cell markers. CSM cells showing a myogenic-like phenotype were also demonstrated through lipid droplets, which were confirmed by Nile Red staining. This cell line was devoid of mycoplasma infection. Inducing the foreign gene plasmid pEGFP-N1 resulted in a 16% transfection efficiency. Sterilized biocompatible seaweed biofilm was employed as a scaffold to grow micro-tissue using the developed CSM cell line. After 20 days of growth, this cell line produced micro-tissue on the seaweed biofilm. Seaweed biofilm is a one-of-a-kind biomaterial that has a wide range of biomedical applications, is ecologically friendly, biocompatible, and helps to improve cellular aquaculture and sustainable development.

China harbors extensive saline-alkaline water resources with considerable potential for aquaculture development. However, their utilization is constrained by high pH, elevated carbonate alkalinity, and complex ionic composition. Exopalaemon carinicauda, a commercially important shrimp species in China, is recognized for its environmental adaptability, rapid growth, desirable flesh quality, and high economic value. Owing to its resilience to diverse environments, E. carinicauda serves as an ideal model for investigating the molecular mechanisms underlying saline-alkaline adaptation in crustaceans. Yet, the genetic determinants of its alkalinity tolerance remain poorly understood, hindering selective breeding efforts. In this study, bulked segregant analysis (BSA) coupled with next-generation sequencing was employed to identify single nucleotide polymorphisms (SNPs) associated with alkalinity tolerance. DNA from individuals exhibiting extreme phenotypes was pooled, and allelic differences were assessed using Euclidean distance, deep learning, and ΔSNP-index methods. A total of 20,879,626 SNPs were detected, and seven candidate genomic regions spanning 47.53 Mb on chromosomes 4, 11, 13, 18, 26, and 36 were identified, encompassing 194 genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed significant associations with GABAergic synapse, taste transduction, and vasopressin-regulated water reabsorption. Comparative transcriptomic analysis under high-alkalinity stress in hepatopancreas and gills identified 28 genes strongly linked to alkalinity tolerance, including gamma-aminobutyric acid receptor, glutamate receptor ionotropic, and basic salivary proline-rich protein. Two SNP loci, C4-2601 and C8-6550, significantly associated with alkalinity tolerance were validated through PCR-based sequencing. These findings provide critical insights into the genetic architecture of alkalinity tolerance in E. carinicauda, facilitating future genomic and marker-assisted selection strategies.

The black rockfish (Sebastes schlegelii), an economically important ovoviviparous teleost in Chinese aquaculture, exhibits a distinctive reproductive strategy characterized by internal fertilization and prolonged embryonic development within females until live birth. However, this reproductive mode poses substantial challenges in artificial breeding practices due to a poor understanding of pregnancy (hereafter referring to intraovarian gestation in S. schlegelii) maintenance mechanisms. Here, we conducted a comparative genomic analysis between S. schlegelii and representative oviparous and ovoviviparous fishes, identifying 374 lineage-specific gene families associated with cellular signaling, metabolic regulation, immune response, and developmental processes. In addition, 73 gene families with significant expansions were identified to be mainly involved in extracellular matrix (ECM) structure and regulation, and neuromodulation. Furthermore, 164 genes potentially associated with mitochondrial function, metabolic pathways and angiogenesis were subjected to strong positive selection. Notably, expression profiles of candidate functional genes associated with ECM structure and regulation, and angiogenesis were detected using transcriptomic data of stage V ovary, optic vesicle stage, and pigmentation stage for ovarian stroma tissue, highlighting the vital roles of ECM remodeling and angiogenesis during pregnancy. These findings provide novel insights into the molecular mechanisms underlying pregnancy maintenance in S. schlegelii and contribute to a broader understanding of the genetic adaptations with pregnancy in ovoviviparous teleosts.

Mollusks are an abundant group of animals, with many ecologically and economically important members that are phylogenetically distinct from nearly all genetic model organisms. This study provides a clade-wide evaluation of small RNA biogenesis pathways, with emphasis on the eastern oyster, Crassostrea virginica. A more thorough characterization of these molecules supports rationale design of RNA interference (RNAi) approaches for manipulation of mollusk genetics. Like other animal groups, mollusks have conserved microRNAs (miRNAs), with some shared with ecdysozoans and deuterostomes; however, there was no evidence of a dedicated endogenous small-interfering RNA (siRNA) pathway. These findings suggest that alternatives to long double-stranded RNA (dsRNA)-mediated knockdown, specifically short-hairpin RNAs or small duplex RNAs, are likely more appropriate for gene silencing in mollusks. The study also finds abundant Piwi-interacting RNAs (piRNAs) in both soma and gonads with some mollusk-specific aspects. Many invertebrates exhibit somatic piRNAs; however, mollusk piRNAs appear to be restricted to a subset of cells, suggesting that the potential of piRNA-based RNAi is also limited. Further, individual animals also express a unique collection of piRNAs that seem to be only partially determined through inheritance from parents. Together, this work defines the RNAi mechanisms in mollusks and provides insights into the phenotypic diversity seen in this group.

Using vacant saline-alkali land for aquaculture has the advantages of resource utilization and significant economic and ecological benefits, but there is a need to understand the impact of variable alkalinity on aquacultural species. This study investigated the impact of different alkaline stress conditions (10 and 20 mmol/L) on transcription and changes in intestinal microbial communities in the oriental river prawn, Macrobrachium nipponense, over a 96-h period. Under low alkalinity conditions, pathways related to carbohydrate metabolism were activated, including glycolysis/gluconeogenesis, mannose metabolism, ascorbate and aldarate metabolism, carbohydrate binding, chitinase activity, and lysosome. Such conditions also led to an increase in the number of beneficial intestinal bacteria, such as Proteobacteria, Firmicutes, Actinobacteriota, and Acidobacteriota. However, high-alkaline conditions inhibited the fibroblast growth factor receptor signaling pathway, store-operated calcium channel activity, and MAPK signaling pathway, and significantly increased the number of pathogenic intestinal bacteria, such as Citrobacter. These results suggest that low alkalinity would promote the growth of M. nipponense by activating the glycolysis pathway and increasing the number of beneficial bacteria. By contrast, high alkalinity would inhibit their immune performance by affecting key signal transduction pathways and increasing harmful bacteria in the intestinal tract. Such insights provide a theoretical basis for the subsequent adaptive aquaculture of M. nipponense in saline-alkali areas.

Sea cucumber Apostichopus japonicus is one of the key aquaculture species in China, possessing high nutritional and medicinal value. However, significant variations in growth are commonly observed among individuals during their development. This study investigates the differences in gut microbiota composition, digestive capacity, and metabolic pathways in A. japonicus, aiming to elucidate the underlying relations contributing to growth rate discrepancies from the perspective of gut microbiota. Intestinal tissues were collected from individuals exhibiting marked growth differences, but Sharing the same genetic background And growth environment, for 16S rRNA sequencing analysis. Although no significant differences were observed in alpha diversity at the ASV level, differences in phylum level proportions suggest potential functional variations. We found significant differences in the gut microbiota between fast-growing and slow-growing A. japonicus by LEfSe analysis. The abundance of fast-growing individuals in Verrucomicrobiota, Verrucomicrobiaceae, and Haloferula was significantly increased. Additionally, significant differences in the enrichment of gut microbiota metabolic pathways were observed; the fast-growing group demonstrated higher overall metabolic activity, and body weight showed a significant positive correlation with glyoxylate and dicarboxylate metabolism and purine metabolism. The digestive capacity of fast-growing A. japonicus was significantly enhanced. Furthermore, fast-growing individuals exhibit a more complex metabolic network involving various biomolecular synthesis and metabolic pathways. These findings suggest that the differences in the growth rate of A. japonicus are primarily related to the functional activity of the gut microbiota; in particular, the improvement of digestive capacity and the activation of specific metabolic pathways may play a key role in promoting the growth of the host. Understanding these microbial influences provides valuable insights into the biological mechanisms underlying growth variation and may contribute to the development of strategies for optimizing sea cucumber aquaculture.

Fish epithelial surfaces are covered by a mucus layer. The highly glycosylated proteins called mucins are a main component of the mucus, which also contains a range of antibacterial enzymes, proteins, and peptides of importance for its protective properties. Here, we compared the practicality and yield of mucus harvesting from barramundi and Atlantic salmon epithelial sites using glass slide, swab, Super·SAL™ and whole tissue extract. We also compared the feasibility of using the orcinol assay, a glycan-on-membrane assay, and absorbance at 230 nm in combination with standard curves of pig gastric mucin to estimate the mucin concentration. Glycomics demonstrated that non-amine hexose content differed more between fish and tissues than terminal monosaccharides with cis-hydroxy groups, and that non-mucin molecules had a major impact on the A230-based results, making the glycan-on-membrane assay the most versatile method for estimating mucin concentration. We conclude that the most versatile tool for mucus harvesting was swabs, allowing for sufficient amounts of sample to be harvested with relative ease and low levels of contamination from the oral cavity, gill, skin, and intestine. Furthermore, the glycan-on-membrane assay was useful for measuring mucus concentration, and it was beneficial to estimate both sample concentration and purity by comparing samples at relatively similar concentrations.