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Ayu (Plecoglossus altivelis) is a commercially important common osmeroid fish for freshwater fisheries. But little is known about the sex-determination (SD) mechanisms. Type IIB endonucleases restriction-site associated DNA sequencing (2b-RAD-seq) is a popular reduced representation method and shown to be an efficient and valuable way for identifying the sex-specific DNA fragments and SD systems. In this study, one male and one female were first whole-genome sequenced. The total size of high-quality genome assembly of the male was 434 Mb (14,900 scaffolds) and of the female 424 Mb (15,340 scaffolds). And ten males and ten females were then subjected to 2b-RAD-seq and isolated 124 candidate male-specific tags and no female-specific tags. The candidate male-specific tags were mapped to 14 male scaffolds, nine of which were aligned to the female allelic scaffolds, and the other five belonged to the candidate male-specific scaffolds. Thereinto, scaf162525, scaf184183, scaf192610 encoded 45 candidate genes in all, 9 of which had functional descriptions. Furthermore, the anti-Mullerian hormone type-2 receptor on scaf192610 was tentatively identified as a Y-linked duplicate copy (Amhr2by) of the autosomal counterpart (Amhr2a), demonstrating the potential role in the SD system of ayu. Based on the four selected male-specific loci, four sets of primers were designed to develop the multiplex-PCR assay. Results showed that the four male-specific markers could identify the genetic sex with 100% accuracy in 126 farmed mature ayu of the different source populations. In conclusion, the genome sequences from both sexes combined with 2b-RAD sequencing analyses from other males and females led to the isolation of male-specific scaffolds, supporting an XX/XY SD system in ayu. The multiplex-PCR assay based on male-specific markers can be applied to perform molecular sexing in ayu culture.

Rapid, sensitive, and specific detection of pathogens is important for effective disease control in aquaculture. However, the use of conventional microbiological methods for pathogen detection is often limited by the time required to complete the assays. The aim of this study was to develop a DNA microarray assay to detect 8 species of pathogenic bacteria, i.e., Vibrio harveyi , V . alginolyticus , V . parahaemolyticus , V . anguillarum , V . cholerae , Nocardia seriolae , Aeromonas hydrophila , and Streptococcus iniae , in cultured aquatic animals. Probes were designed according to a variable region of the bacterial 16S rRNA gene and gryB sequences, and 23 probes were finally selected by hybridization with 25 bacterial type cultures. The microarray sensitivity for V. anguillarum detection in healthy fish blood was ≥ 100 CFU/mL. Pathogenic bacteria in blood samples from 21 diseased aquacultural samples were analyzed in a blinded fashion by using the microarray, conventional culture, and 16S rRNA gene sequencing methods; the results of the microarray assay agreed well with those of the conventional culture and 16S rRNA gene sequencing methods. The results of this study show that the microarray method described is suitable for preliminary diagnoses or for confirmation of the main potential pathogens in aquatic animals.