Diversity and Antimicrobial Activity of Intestinal Fungi from Three Species of Coral Reef Fish.

Although intestinal microbiota play crucial roles in fish digestion and health, little is known about intestinal fungi in fish. This study investigated the intestinal fungal diversity of three coral reef fish (Lates calcarifer, Trachinotus blochii, and Lutjanus argentimaculatus) from the South China Sea using a culturable method. A total of 387 isolates were recovered and identified by sequencing their internal transcribed spacer sequences, belonging to 29 known fungal species. The similarity of fungal communities in the intestines of the three fish verified that the fungal colonization might be influenced by their surrounding environments. Furthermore, the fungal communities in different intestines of some fish were significantly different, and the number of yeasts in the hindgut was less than that in fore- and mid-intestines, suggesting that the distribution of fungi in fishes' intestines may be related to the physiological functions of various intestinal segments. In addition, 51.4% of tested fungal isolates exhibited antimicrobial activity against at least one marine pathogenic microorganism. Notably, isolate Aureobasidium pullulans SCAU243 exhibited strong antifungal activity against Aspergillus versicolor, and isolate Schizophyllum commune SCAU255 displayed extensive antimicrobial activity against four marine pathogenic microorganisms. This study contributed to our understanding of intestinal fungi in coral reef fish and further increased the library of fungi available for natural bioactive product screening.

Oil-contaminated sites act as high-risk pathogen reservoirs previously overlooked in coastal zones.

In addition to the organic pollutants and disturbance to the microbial, plant and animal systems, oil contamination can also enrich opportunistic pathogens. But little is known about whether and how the most common coastal oil-contaminated water bodies act as reservoirs for pathogens. Here, we delved into the characteristics of pathogenic bacteria in coastal zones by constructing seawater-based microcosms with diesel oil as a pollutant. 16S rRNA gene full-length sequencing and genomic exploration revealed that pathogenic bacteria with genes involved in alkane or aromatic degradation were significantly enriched under oil contamination, providing a genetic basis for them to thrive in oil-contaminated seawater. Moreover, high-throughput qPCR assays showed an increased abundance of the virulence gene and enrichment in antibiotics resistance genes (ARGs), especially those related to multidrug resistance efflux pumps, and their high relevance to Pseudomonas, enabling this genus to achieve high levels of pathogenicity and environmental adaptation. More importantly, infection experiments with a culturable P. aeruginosa strain isolated from an oil-contaminated microcosm provided clear evidence that the environmental strain was pathogenic to grass carp (Ctenopharyngodon idellus), and the highest lethality rate was found in the oil pollutant treatment, demonstrating the synergistic effect of toxic oil pollutants and pathogens on infected fish. A global genomic investigation then revealed that diverse environmental pathogenic bacteria with oil degradation potential are widely distributed in marine environments, especially in coastal zones, suggesting extensive pathogenic reservoir risks in oil-contaminated sites. Overall, the study uncovered a hidden microbial risk, showing that oil-contaminated seawater could be a high-risk pathogen reservoir, and provides new insights and potential targets for environmental risk assessment and control.

Characterization of the complete mitochondrial genome of blacktip shark Carcharhinus limbatus (Carcharhiniformes: Carcharhinidae).

In this study, we aimed to determine the complete mitochondrial genome of blacktip shark Carcharhinus limbatus. The mitochondrial genome was 16,705 bp in length, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region. Phylogenetic analysis was done using the Bayesian inference method, which showed a close relationship between C. limbatus and C. amblyrhynchoides.

Rapid and Visual Detection of Vibrio parahaemolyticus in Aquatic Foods Using blaCARB-17 Gene-Based Loop-Mediated Isothermal Amplification with Lateral Flow Dipstick (LAMP-LFD).

Vibrio parahaemolyticus is recognized as one of the most important foodborne pathogens responsible for gastroenteritis in humans. The blaCARB-17 gene is an intrinsic ß-lactamase gene and a novel species-specific genetic marker of V. parahaemolyticus. In this study, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) was developed targeting this blaCARB-17 gene. The specificity of LAMP-LFD was ascertained by detecting V. parahaemolyticus ATCC 17802 and seven other non-V. parahaemolyticus strains. Finally, the practicability of LAMP-LFD was confirmed by detection with V. parahaemolyticus-contaminated samples and natural food samples. The results showed that the optimized reaction parameters of LAMP are as follows: 2.4 mmol/l Mg2+, 0.96 mmol/l dNTPs, 4.8 U Bst DNA polymerase, and an 8:1 ratio of inner primer to outer primer, at 63°C for 40 min. The optimized reaction time of the LFD assay is 60 min. Cross-reactivity analysis with the seven non-V. parahaemolyticus strains showed that LAMP-LFD was exclusively specific for V. parahaemolyticus. The detection limit of LAMP-LFD for V. parahaemolyticus genomic DNA was 2.1 × 10-4 ng/µl, corresponding to 630 fg/reaction and displaying a sensitivity that is 100-fold higher than that of conventional PCR. LAMP-LFD in a spiking study revealed a detection limit of approximately 6 CFU/ml, which was similar with conventional PCR. The developed LAMP-LFD specifically identified the 10 V. parahaemolyticus isolates from 30 seafood samples, suggesting that this LAMP-LFD may be a suitable diagnostic method for detecting V. parahaemolyticus in aquatic foods.

[Sited-directed mutagenesis of hCu, Zn-SOD gene and its expression in Synechococcus sp. PCC7942].

The Cys111 genetic code of human copper/zinc superoxide dismutase (hCu, Zn-SOD) gene in the pESOD plamid was mutated into the Ala111 code with site-directed mutagenesis, and then the plamid pESODT111 which contained groESL promoter, mutated hCu, Zn-SOD gene, rbcS-polyA terminator and reporter gene (Kanr) was constructed and transduced into Synechococcus sp. PCC7942 with homologous recombination platform. The results of PCR and DNA sequence analysis showed that the target nucleotide had been genetically integrated into genome DNA of the host cell. SDS-PAGE, Western blot and Pyrogallol autoxidation assay confirmed that the transformant strains expressed the mutated hCu, Zn-SOD protein. And the level of the mutated hCu, Zn-SOD protein reached a value of 3.61% of the total soluble protein. Furthermore, the transformants still retained 95% activities of SOD after 30 minutes at 80 degrees C environment, it indicated that the mutated hCu, Zn-SOD protein could endure higher temperature than the natural one.

Complete mitochondrial genome of Chinese lake gudgeon Sarcocheilichthys sinensis (Cypriniformes, Cyprinidae).

The complete mitochondrial genome of the Chinese lake gudgeon Sarcocheilichthys sinensis was first determined in this study. It is a circular DNA double strand of 16,684 bp in length, encodes genes for 13 proteins, 2 ribosomal RNA subunits, 22 transfer RNAs and an A + T-rich control region with the typical gene order in vertebrate mitogenomes. Overall nucleotide composition is 30.5% A, 26.6% C, 16.7% G and 26.3% T. Three start codons (ATG, GTG and ATA) and three stop codons (TAG, TAA and T) were found in all protein-coding genes. The tRNA-Ser(AGY) lacked the dihydrouridine arm and could not fold into typical cloverleaf secondary structure. The origin of L-strand replication was identified between the tRNA-Asn and tRNA-Cys genes.