A modified genomic DNA extraction method named the combination of lysozyme and ultrasonic lysis (CLU) method was used to analyze the fish intestinal microflora. In this method, the physical disruption and chemical lysis steps were combined, and some parameters in the key steps were adjusted. In addition, the results obtained by this method were compared with the results obtained by the Zirmil-beating cell disruption method and the QIAamp Fast DNA Stool Mini Kit. The OD260/OD280ratio and concentration of the DNA extracted using the CLU method were 2.02 and 282.8 µg/µL, respectively; when the incubation temperatures for lysozyme and RNase were adjusted to 37 °C, those values were 2.08 and 309.8 µg/µL, respectively. On the agarose gel, a major high-intensity, discrete band of more than 10 kb was found for the CLU method. However, the smearing intensity of degraded DNA was lower when the incubation temperatures were 60 °C for lysozyme and 30 °C for RNase than when incubation temperatures of 37 °C for lysozyme and 37 °C for RNase were used. The V3 variable region of the prokaryotic 16S rDNA was amplified, and an approximately 600-bp fragment was observed when the DNA extracted using the CLU method was used as a template. The CLU method is simple and cost effective, and it yields high-quality, unsheared, high-molecular-weight DNA, which is comparable to that obtained with a commercially available kit. The extracted DNA has potential for applications in critical molecular biology techniques.
To better understand gene expression in the intestine after Shewanella algae infection and provide insights into its immune roles in the tongue sole, Cynoglossus semilaevis, sequencing-based high-throughput RNA analysis (RNA-Seq) for the intestines between the control group and 12 h post-injection group was performed. After assembly, there was an average of 23,957,159 raw sequencing reads, and 23,943,491 clean reads were obtained after filtering out low-quality reads. Then, 383 differentially expressed genes (DEGs) in the intestines in response to S. algae infection were identified. Subsequently, gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs were conducted to further explore their functions. Among all of the pathways involved, sixteen pathways were related to the immune system, among which the complement and coagulation cascades pathway was the most prominent for immunity-related DEGs, followed by the leukocyte transendothelial migration pathway. Furthermore, the expression levels of twelve selected DEGs in the immune-related pathways were identified by quantitative real-time polymerase chain reaction, substantiating the reliability and reproducibility of the RNA-Seq results. In summary, this study represents an important genomic resource for understanding the potential immune role of the tongue sole intestine from the perspective of gene expression.
Two DNA extraction methods, the Zirmil-beating cell disruption method (ZBC) and the QIAamp fast DNA stool mini kit (QIA), were used to extract DNA from the intestinal flora of the penaeid shrimp Litopenaeus vannamei, and their microbial communities were analyzed using 16S rDNA high-throughput sequencing. Results were obtained in terms of the number of reads, alpha diversity indexes, beta diversity indexes and taxonomic composition. The alpha diversity indexes of the community, according to the ZBC method, were higher than those according to the QIA method. Furthermore, results from the three samples using the ZBC method were less consistent than those where the QIA method was used. Further, using the latter method led to substantive clustering. It is suggested that the QIA method is more stable and repeatable than the ZBC method. Although the two extraction methods shared the major abundant microflora based on 16S rDNA high-throughput sequencing, bias associated with diversity analysis indexes and certain species was observed.
The virulence of Vibrio parahaemolyticus is variable depending on its virulence determinants. A V. parahaemolyticus strain, in which the virulence is governed by the pirA and pirB genes, can cause acute hepatopancreatic necrosis disease (AHPND) in shrimps. Some V. parahaemolyticus that are non-AHPND strains also cause shrimp diseases and result in huge economic losses, while their pathogenicity and pathogenesis remain unclear. In this study, a non-AHPND V. parahaemolyticus, TJA114, was isolated from diseased Penaeus vannamei associated with a high mortality. To understand its virulence and adaptation to the external environment, whole-genome sequencing of this isolate was conducted, and its phenotypic profiles including pathogenicity, growth characteristics and nutritional requirements were investigated. Shrimps following artificial infection with this isolate presented similar clinical symptoms to the naturally diseased ones and generated obvious pathological lesions. The growth characteristics indicated that the isolate TJA114 could grow well under different salinity (10-55 p.p.t.), temperature (23-37 °C) and pH (6-10) conditions. Phenotype MicroArray results showed that this isolate could utilize a variety of carbon sources, amino acids and a range of substrates to help itself adapt to the high hyperosmotic and alkaline environments. Antimicrobial-susceptibility test showed that it was a multidrug-resistant bacterium. The whole-genomic analysis showed that this V. parahaemolyticus possessed many important functional genes associated with multidrug resistance, stress response, adhesions, haemolysis, putative secreted proteases, dedicated protein secretion systems and a variety of nutritional metabolic mechanisms. These annotated functional genes were confirmed by the phenotypic profiles. The results in this study indicated that this V. parahaemolyticus isolate possesses a high pathogenicity and strong environmental adaptability.