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Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10-1 pg/μl for genomic DNA of tested bacteria and 10-4 to 10-5 pg/μl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.

A 20-day trial was conducted to reveal bacterial community dynamics in a commercial nursery of larval Litopenaeus vannamei larvae. The bacterial communities in the ambient water were profiled by high-throughput sequencing of the V4-V5 hypervariable region of the 16S rRNA gene. The results indicated that the dominant bacterial phyla between the metamorphosis stage and postlarval stage were Bacteroidetes, Proteobacteria, Cyanobacteria, and Firmicutes, representing more than 80.09% of the bacterial operational taxonomic units. The relative abundance among bacterial phyla notably differed between the two stages. The relative abundance of Cyanobacteria was higher in the metamorphosis stage, while that of Bacteroidetes was higher and more stable in the postlarval stage. At the class level, the relative abundance of Sphingobacteriia and Alphaproteobacteria increased markedly in the postlarval stage, while that of Flavobacteriia decreased. Redundancy analysis showed that bacterial composition in the metamorphosis stage was positively correlated with salinity, alkalinity, and pH, while in the postlarval stage, it was positively correlated with ammonium nitrogen and nitrite nitrogen. Thus, microbial community diversity in the nursery phase varies per rearing stage.

Immune response of three crustin-like genes isolated from Litopenaeus vannamei under Vibrio harveyi infection was analyzed. Sequence analysis revealed that three crustin-like proteins contained whey acidic protein (WAP) domain, Crs 1 encoded 126 amino acids, Crs 2 encoded 156 amino acids, and Crs3 encoded 87 amino acids. Tissue expression pattern analysis showed that Crs 1 was not detected in the intestine, it was highest expressed in the muscle and lowest expressed in the hepatopancreas; Crs 2 was not detected in the hepatopancreas, intestine, and muscle but it was highest expressed in the hemocyte. Crs 3 was expressed in all tested tissues and was more abundant in the gill and muscle. Bacterial infection assay showed that mRNA level of Crs 1 in the gill was significantly upregulated at 24 h and 7 days; it was upregulated at 7 days in the muscle. The transcript level of Crs 2 in the hemocyte was upregulated at 24 h and significantly downregulated at 7 days, and it was downregulated in the heart at 24 h and 7 days. mRNA levels of Crs 3 in the hemocyte and hepatopancreas were significantly increased at 24 h; it was upregulated at 7 days in the heart and muscle. The results indicated that immune response of different crustins was tissue specific and the data provided us basic knowledge about the roles of three crustin-like genes in response to bacterial infection.

Waterborne pathogens are becoming a serious worldwide health hazard; thus, the regular monitoring of epidemic pathogens is urgently required for public safety. In the present study, we developed a microfluidic chip integrated loop-mediated isothermal amplification technique (on-chip LAMP) to simultaneously detect 10 waterborne pathogenic bacteria, Campylobacter jejuni, Listeria monocytogenes, Salmonella enterica, Shigella flexneri, Staphylococcus aureus, Vibrio alginolyticus, V. cholerae, V. parahemolyticus, V. vulnificus, and Yersinia enterocolitica. This method was capable of simultaneously completing 22 genetic analyses of two specimens and achieved limits of detection ranging from 7.92 × 10-3 to 9.54 × 10-1 pg of genomic DNA of pure bacteria per reaction. The processes from sample loading to microfluidic operation were in a highly automated format, and the LAMP reaction ran to completion within 35 minutes, with a minimal volume of 22 μl per each half of a single chip. The coefficient of variation for the time-to-positive value was less than 0.1, indicating an excellent reproducibility of the dual-sample on-chip LAMP assay. The clinical sensitivity and specificity in analyses of coastal water samples were 93.1% and 98.0%, respectively, in comparison with traditional microbiological methods. Our established dual-sample on-chip LAMP assay provides an effective multiple-pathogen analysis of waterborne bacterial pathogens. This indicates that the method is applicable for on-site detection and routine monitoring of waterborne bacteria in aquatic environments.

White spot syndrome virus (WSSV), a double-stranded DNA virus that infects crustaceans, is the most serious viral pathogen affecting shrimp farming worldwide. To reduce the economic losses caused by WSSV, we screened a novel coumarin derivative from a small molecule drug library, N-(4-((4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)sulfonyl)phenyl)acetamide (N2905), to evaluate its anti-WSSV effects in vivo. We determined that compound N2905, up to a concentration of 20 mg/L, significantly decreased the number of WSSV copies in Litopenaeus vannamei post-larvae, with a maximum inhibitory rate of > 90 %, and increased the survival rate of WSSV-infected post-larvae. Pre-treatment and post-treatment assays indicated that N2905 could treat, but not prevent, WSSV infections. When WSSV was preincubated with N2905 for 1-4 h, the incidence of viral infections was significantly reduced and survival time of post-larvae extended to 120 h. A stability study of N2905 provided a reference for its practical use. Considering the antiviral stability of N2905 in culture water within 2 d, continuous N2905 exchange was performed, showing a significant decrease in viral load at 120 h post-infection (hpi) and a 55 % increase in survival of WSSV-infected post-larvae. Overall, our study demonstrated the potential of N2905 as an antiviral agent.

The complete mitochondrial genome sequence of Metapenaeus ensis was obtained by PCR, containing 37 genes with 13 protein-coding genes, 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs) and a non...