Marine Biotechnology最新文献

The vitellogenin present in the bloodstream undergoes internalization into developing oocytes through the vitellogenin receptor (VgR), a process mediated by receptor-mediated endocytosis. VgR plays a crucial role in facilitating the accumulation of vitellogenin and the maturation of oocytes. In this study, we characterized a Tachypleus tridentatus vitellogenin receptor (TtVgR) gene from the tri-spine horseshoe crab, revealing a length of 1956 bp and encoding 652 amino acid residues with 12 exons. TtVgR has a molecular weight of 64.26 kDa and an isoelectric point of 5.95. Predictions indicate 85 phosphorylation sites and 7 glycosylation sites within TtVgR. Transcriptional analysis demonstrated specific expression of TtVgR in the ovary and yellow connective tissue. TtVgR was identified and distributed in the plasma membrane of oocytes. The siRNA-mediated TtVgR knockdown significantly reduced the transcriptional activity of TtVgR. This depletion induced excessive ROS production, resulting in DNA damage in ovarian primary cells. TUNEL and flow cytometry analyses confirmed ovarian cell apoptosis following TtVgR knockdown, indicating DNA damage in ovarian primary cells. These findings underscore the importance of TtVgR in ovarian cell development, suggesting its potential involvement in vitellogenesis and oocyte maturation. This knowledge may inform innovative breeding strategies and contribute to the sustainable management and conservation of the tri-spine horseshoe crab.

Polysaccharide-degrading bacteria are key participants in the global carbon cycle and algal biomass recycling. Herein, a polysaccharide lyase-producing strain HB226069 was isolated from Sargassum sp. from Qingge Port, Hainan, China. Results of the phylogenetic of the 16S rRNA gene and genotypic analysis indicated that the isolate should be classified as Microbulbifer thermotolerans. The whole genome is a 4,021,337 bp circular chromosome with a G+C content of 56.5%. Analysis of the predicted genes indicated that strain HB226069 encoded 161 carbohydrate-active enzymes (CAZymes), and abundant putative enzymes involved in polysaccharide degradation were predicted, including alginate lyase, fucosidase, agarase, xylanase, cellulase, pectate lyase, amylase, and chitinase. Three of the putative polysaccharide lyases from PL7 and PL17 families were involved in alginate degradation. The alginate lyases of strain HB226069 showed the maximum activity of 117.4 U/mL at 50 °C, pH 7.0, and 0.05 M FeCl₃, while exhibiting the best stability at 30 °C and pH 7.0. The Thin Layer Chromatography (TLC) and Electrospray Ionization Mass Spectrometry (ESI-MS) analyses indicated that the alginate oligosaccharides (AOSs) degraded by the partially purified alginate lyases contained oligosaccharides of DP2–DP5 and monosaccharide while reacting for 36 h. The complete genome of M. thermotolerans HB226069 enriches our understanding of the mechanism of polysaccharide lyase production and supports its potential application in polysaccharide degradation.

Many organisms incorporate inorganic solids into their tissues to improve functional and mechanical properties. The resulting mineralized tissues are called biominerals. Several studies have shown that nacreous biominerals induce osteoblastic extracellular mineralization. Among them, Pinctada margaritifera is well known for the ability of its organic matrix to stimulate bone cells. In this context, we aimed to study the effects of shell extracts from three other Pinctada species (Pinctada radiata, Pinctada maxima, and Pinctada fucata) on osteoblastic extracellular matrix mineralization, by using an in vitro model of mouse osteoblastic precursor cells (MC3T3-E1). For a better understanding of the Pinctada-bone mineralization relationship, we evaluated the effects of 4 other nacreous mollusks that are phylogenetically distant and distinct from the Pinctada genus. In addition, we tested 12 non-nacreous mollusks and one extra-group. Biomineral shell powders were prepared, and their organic matrix was partially extracted using ethanol. Firstly, the effect of these powders and extracts was assessed on the viability of MC3T3-E1. Our results indicated that neither the powder nor the ethanol-soluble matrix (ESM) affected cell viability at low concentrations. Then, we evaluated osteoblastic mineralization using Alizarin Red staining and we found a prominent MC3T3-E1 mineralization mainly induced by nacreous biominerals, especially those belonging to the Pinctada genus. However, few non-nacreous biominerals were also able to stimulate the extracellular mineralization. Overall, our findings validate the remarkable ability of CaCO₃ biomineral extracts to promote bone mineralization. Nevertheless, further in vitro and in vivo studies are needed to uncover the mechanisms of action of biominerals in bone.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely applied in animals as an efficient genome editing tool. However, the technique is difficult to implement in fish cell lines partially due to the lack of efficient promoters to drive the expression of both sgRNA and the Cas9 protein within a single vector. In this study, it was indicated that the zebrafish U6 RNA polymerase III (ZFU6) promoter could efficiently induce tyrosinase (tyr) gene editing and lead to loss of retinal pigments when co-injection with Cas9 mRNA in zebrafish embryo. Furthermore, an optimized all-in-one vector for expression of the CRISPR/Cas9 system in the zebrafish fibroblast cell line (PAC2) was constructed by replacing the human U6 promoter with ZFU6 promoter, basing on the lentiCRISPRV2 system that widely applied in mammal cells. This new vector could successfully target the cellular communication network factor 2a (ctgfa) gene and demonstrated its function in the PAC2 cell. Notably, the vector could also be used to edit the endogenous EMX1 gene in the mammal 293 T cell line, implying its wide application potential. In conclusion, we established a new gene editing tool for zebrafish cell line, which could be a useful in vitro platform for high-throughput analyzing gene function in fish.

This study is the first investigation for using sex-related gene expression in tail fin tissues of seabass as early sex determination without killing the fish. The European seabass (Dicentrarchus labrax) is gonochoristic and lacks distinguishable sex chromosomes, so, sex determination is referred to molecular actions for some sex-related genes on autosomal chromosomes which are well known such as cyp19a1a, dmrt1a, and dmrt1b genes which play crucial role in gonads development and sex differentiation. cyp19a1a is expressed highly in females for ovarian development and dmrt1a and dmrt1b are for testis development in males. In this study, we evaluated the difference in the gene expression levels of studied genes by qPCR in tail fins and gonads. We then performed discriminant analysis (DA) using morphometric traits and studied gene expression parameters as predictor tools for fish sex. The results revealed that cyp19a1a gene expression was significantly higher in future females’ gonads and tail fins (p ≥ 0.05). Statistically, cyp19a1a gene expression was the best parameter to discriminate sex even the hit rate of any other variable by itself could not correctly classify 100% of the fish sex except when it was used in combination with cyp19a1a. In contrast, Dmrt1a gene expression was higher in males than females but there were difficulties in analyzing dmrt1a and dmrt1b expressions in the tail because levels were low. So, it could be used in future research to differentiate and determine the sex of adult fish using the cyp19a1a gene expression marker without killing or sacrificing fish.

This study assessed the effects of dietary supplementation of poly-β-hydroxybutyrate (PHB) on growth performance, feed efficiency, non-specific immunity, digestive enzyme capacity, phagocytic activity, hemocyte count, intestinal morphology, and disease resistance against Vibrio parahaemolyticus of Pacific white shrimp (Penaeus vannamei). Six diets were prepared by supplementing graded levels of PHB at 0.00, 0.25, 0.50, 1.00, 2.00, and 4.00% (Con, P0.25, P0.5, P1.0, P2.0, and P4.0, respectively). Triplicate groups of 90 shrimps (initial body weight 0.25 ± 0.01 g) per treatment were randomly assigned and fed an experimental diet for 56 days. The growth performance of shrimp was significantly improved by 1% dietary PHB supplementation. PHB-included diets fed shrimp showed significantly improved hepatopancreatic trypsin, chymotrypsin, and pepsin activities. Villus height was significantly increased with dietary PHB supplementation, and villus width was increased at a 1% inclusion level. P0.25, P0.5, and P4.0 groups significantly increased phenoloxidase activity, and the P2.0 group significantly increased anti-protease activity compared to the Con group. The survival of shrimp challenged against V. parahaemolyticus was higher in P0.5, P1.0, and P2.0 groups than in the Con diet. Dietary PHB supplementation improved weight gain, digestive enzyme activity, intestinal morphology, non-specific immunity, and disease resistance against V. parahaemolyticus of shrimp. According to the above observations, the optimal dietary PHB supplementation level for maximum weight gain would be 1% for Pacific white shrimp.

High-temperature stress poses a significant environmental challenge for aquatic organisms, including tsinling lenok trout (Brachymystax lenok tsinlingensis). This study aimed to investigate the role of microRNAs (miRNAs) in inducing liver inflammation in tsinling lenok trout under high-temperature stress. Tsinling lenok trout were exposed to high-temperature conditions (24 °C) for 8 h, and liver samples were collected for analysis. Through small RNA sequencing, we identified differentially expressed miRNAs in the liver of high-temperature-stressed tsinling lenok trout compared to the control group (maintained at 16 °C). Several miRNAs, including novel-m0105-5p and miR-8159-x, showed significant changes in expression levels. Additionally, we conducted bioinformatics analysis to explore the potential target genes of these differentially expressed miRNAs. Our findings revealed that these miRNA target genes are involved in inflammatory response pathways, such as NFKB1 and MAP3K5. The downregulation of novel-m0105-5p and miR-8159-x in the liver of high-temperature-stressed tsinling lenok trout suggests their role in regulating liver inflammatory responses. To validate this, we performed a dual-luciferase reporter assay to confirm the regulatory relationship between miRNAs and target genes. Our results demonstrated that novel-m0105-5p and miR-8159-x enhance the inflammatory response of hepatocytes by promoting the expression of NFKB1 and MAP3K5, respectively. In conclusion, our study provides evidence that high-temperature stress induces liver inflammation in tsinling lenok trout through dysregulation of miRNAs. Understanding the molecular mechanisms underlying the inflammatory response in tsinling lenok trout under high-temperature stress is crucial for developing strategies to mitigate the negative impacts of environmental stressors on fish health and aquaculture production.

Tetrodotoxin (TTX), known as pufferfish toxin, is a potent neurotoxin blocking sodium channels in muscle and nerve tissues. TTX has been detected in various taxa other than pufferfish, including marine polyclad flatworms, suggesting that pufferfish toxin accumulates in fish bodies via food webs. The composition of TTX and its analogs in the flatworm Planocera multitentaculata was identical to those in wild grass puffer Takifugu alboplumbeus. Previously, Planocera sp. from Okinawa Island, Japan, were reported to possess high level of TTX, but no information was available on TTX analogs in this species. Here we identified TTX and analogs in the planocerid flatworm using high-resolution liquid chromatography–mass spectrometry, and compared the composition of TTX and analogs with those of another toxic and non-toxic planocerid species. We show that the composition of TTX and several analogs, such as 5,6,11-trideoxyTTX, dideoxyTTXs, deoxyTTXs, and 11-norTTX-6(S)-ol, of Planocera sp. was identical to those of toxic species, but not to its non-toxic counterpart. The difference in the toxin composition was reflected in the phylogenetic relationship based on the mitochondrial genome sequence. A toxification experiment using predatory fish and egg plates of P. multitentaculata demonstrated that the composition of TTX and analogs in wild T. alboplumbeus juveniles was reproduced in artificially toxified pufferfish. Additionally, feeding on the flatworm egg plates enhanced the signal intensities of all TTX compounds in Chelonodon patoca and that of deoxyTTXs in Yongeichthys criniger.

Colorful shells in bivalves are mostly caused by the presence of biological pigments, among which melanin is a key component in the formation of shell colours. Cyclic adenosine monophosphate (cAMP) is an important messenger in the regulation of pigmentation in some species. However, the role of cAMP in bivalve melanogenesis has not yet been reported. In this study, we performed in vitro and in vivo experiments to determine the role of cAMP in regulating melanogenesis in Pacific oysters. Besides, the function of cAMP-responsive element modulator (CREM) and the interactions between CREM and melanogenic genes were investigated. Our results showed that a high level of cAMP promotes the expression of melanogenic genes in Pacific oysters. CREM controls the expression of the MITF gene under cAMP regulation. In addition, CREM can regulate melanogenic gene expression, tyrosine metabolism, and melanin synthesis. These results indicate that cAMP plays an important role in the regulation of melanogenesis in Pacific oysters. CREM is a key transcription factor in the oyster melanin synthesis pathway, which plays a crucial role in oyster melanin synthesis through a cAMP-mediated CREM-MITF-TYR axis.

Shell color as an important economic trait is also the crucial target trait for breeding and production. MicroRNA (miRNA) is an endogenous small non-coding RNA that can post-transcriptionally regulate the expression of target genes, it plays important roles in many life activities and physiological processes, such as shell color, stress response, and disease traits. In this study, we investigated the function of lgi-miR-2d in shell melanin formation and the expression patterns of lgi-miR-2d and target gene Rpmitf in Manila clam Ruditapes philippinarum. We further explored and verified the relationship between Rpmitf and lgi-miR-2d and identified the expression level of shell color-related gene changes by RNAi and injecting the antagomir of lgi-miR-2d, respectively. Our results indicated that lgi-miR-2d antagomir affected the expression of its target gene Rpmitf. In addition, the dual-luciferase reporter assay was conducted to confirm the direct interaction between lgi-miR-2d and Rpmitf. The results showed that the expression levels of melanin-related genes such as Rpmitf and tyr were significantly decreased in the positive treatment group compared with the blank control group after the Rpmitf dsRNA injection, indicating Rpmitf plays a crucial role in the melanin synthesis pathway. Taken together, we speculated that lgi-miR-2d might be negatively modulating Rpmitf, which might regulate other shell color-related genes, thereby affecting melanin synthesis in R. philippinarum.