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Coilia nasus is an endangered fish species in the Yangtze River, and there is urgent need to know the genes related to its reproduction and fertility. The DAZ family including boule, daz and dazl, plays an important role in germ cells development. In this study, the cDNA sequence of boule (Cnbol) and dazl (Cndazl) genes were cloned and their expression patterns were characterized in Coilia nasus. RT-PCR showed that the expression of Cnbol and Cndazl mRNAs was restricted in adult gonads. The section in situ hybridization indicated that the temporospatial expression patterns of Cnbol and Cndazl were significantly different. In the testis, Cnbol was mainly detected in spermatocytes and spermatids, while Cndazl was prominently expressed in s permatogonia. In the ovary, Cnbol and Cndazl were highly expressed in the early stages of oocytes. Interestingly, Cndazl was also concentrated in perinuclear speckle and then localized to the Balbiani body in late stages of oocytes. In addition, Cnbol and Cndazl 3′ UTR can specifically label medaka Primordial Germ Cells (PGCs). According to our results, Cnbol and Cndazl are marker genes of germ cells and may play a vital role in the development and differentiation of germ cells in Coilia nasus.

Coilia nasus is a precious migratory fish typical to the Yangtze River Basin of China. It is locally referred to as the "Three delicious fish of the Yangtze River, " with its fresh meat being particularly delicious and popular. In recent years, wild C. nasus numbers in the Yangtze River Basin have been decreasing due to environmental changes and illegal overfishing, among other reasons. To attenuate the availability of C. nasus, the Shanghai Fisheries Research Institute has successfully established an indoor artificial breeding and pond culture program in 2011. The research and development of C. nasus artificial breeding technologies have steadily progressed, though the artificial breeding yield of C. nasus remains relatively low. In addition, related research on the artificial cultivation of C. nasus is still in its infancy. Research on C. nasus in China and abroad mainly focuses on gonad development, breeding technology, muscle nutrient composition, and growth performance. No reports exist on the effects of brackish water culture conditions on the physiology of C. nasus. Given its economic importance, it would be of great value to evaluate the changes in the digestive, antioxidant, nonspecific immune capacity, and metabolic rates of C. nasus from brackish water (natural seawater in Hangzhou Bay, salinity 8.7~12.5) to freshwater aquaculture, to determine the breeding conditions of C. nasus, and to improve the artificial breeding yield and related technology. To investigate the differences in digestive capacity, antioxidant capacity, nonspecific immune capacity, and metabolic rates of C. nasus in brackish water and freshwater aquaculture conditions, the experiment adopted the pond interbreeding method. Two groups were established: A brackish aquaculture group and a freshwater aquaculture group (control group). Each group was housed in a single aquaculture pond with an area of 0.17 hm2. 500 C. nasus were stocked in each pond and moved to a Takifugu obscurus pond for a seven-month aquaculture experiment. At the end of the experiment, 12 C. nasus were randomly selected from each group and randomly divided into three replicates. Four C. nasus samples were selected from each replicate. The liver, intestine, stomach, and cecum of C. nasus were dissected on an ice plate and carefully removed to prepare a 10% homogenate. The activities of digestive enzymes, antioxidant enzymes, nonspecific immune enzymes, and metabolic enzymes and the total protein content were measured using a kit produced by the Nanjing Jiancheng Bioengineering Institute. The activities of digestive enzymes, antioxidant enzymes, nonspecific immunity enzymes, and metabolic enzymes in brackish water and freshwater aquaculture environments were compared and analyzed in a seven-month aquaculture experiment using the same batch of artificially bred C. nasus. The growth and survival rates of C. nasus cultured in brackish water (hereafter referred to as brackish water C. nasus) were significantly higher than that in the freshwater (hereafter referred to as freshwater C. nasus) (P < 0.05). The digestive ability of protein and starch in brackish water C. nasus was higher than that of freshwater C. nasus, while the digestive ability was weakened. Among them, the activity of amylase (AMS) in the four organs of brackish water C. nasus was higher than those in freshwater, but not significantly so (P > 0.05). The activities of protease in the liver and stomach were significantly higher than those in freshwater C. nasus (P < 0.05), and the activities of lipase (LPS) in the liver and cecum were significantly lower than those in freshwater C. nasus (P < 0.05). The order of AMS and protease activity of brackish water C. nasus was: cecum > intestine > stomach > liver, while the order of LPS activity was intestine > cecum > stomach > liver. The order of AMS, protease, and LPS activity of freshwater C. nasus was: cecum > intestine > stomach > liver. Brackish water had significant effects on antioxidant, nonspecific immunity, and metabolic abilities, and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and malondialdehyde (MDA) in the liver of C. nasus were significantly increased, while the activity of catalase (CAT) was significantly decreased (P < 0.05). The activities of alkaline phosphatase (AKP) and acid phosphatase (ACP) in the liver of C. nasus in brackish water were significantly lower than those in freshwater (P < 0.05). Brackish water had no significant effect on the activity of aspartate aminotransferase (AST), but it significantly reduced the activity of alanine aminotransferase (ALT). In summary, brackish water aquaculture conditions promoted the growth of C. nasus; increased the survival rate; improved the ability of C. nasus to digest protein and starch; improved the immunity of fish; and reduced the influence of external stresses by decreasing the activities of AKP, ACP, and ALT enzymes and increasing the activities of SOD and GSH-PX enzymes. This study also found that C. nasus living in brackish water, as a result of maintaining the internal osmotic balance, significantly reduced ALT enzyme activity and reduced urea production and emission, thereby reducing the pollution of the direct environment. Therefore, brackish water is more suitable for the aquaculture of C. nasus, and it is recommended that brackish water (salinity of 8.7~12.5) be used for aquaculture of 1+ age C. nasus individuals in the future. The results of this study provide a scientific basis for the improvement of artificial breeding technology, improving the yield of C. nasus, and the development of special compound feed for C. nasus.

This study was designed to generate the full-length transcriptome of Coilia nasus using single-molecule long read isoform sequencing (SMRT-seq) technology. RNAs of brains, ovaries, and testes of C. nasus conditioned in both seawater and freshwater except for gut which was only from the fish conditioned in freshwater, were isolated. All the RNAs were pooled together equally for SMRT sequencing. One SMRT cell produced 64.18 Gb clean data, including 765,184 circular consensus (CCS) reads and 616,126 full-length non-chimeric (FLNC) reads. Finally, 93,793 non-redundant transcripts were obtained after clustered and polished. Structural analysis of the 93,793 non-redundant transcripts predicted 8242 alternative splicing events, a total of 229,825 SSR and 72,037 complete CDS. A total of 8437 lncRNA were identified. 84,617 transcripts were annotated in NR, Swissprot, GO, COG, KOG, eggnog, Pfam and KEGG database totally. This is the first full-length transcriptome of Coilia nasus, including transcripts appeared in the case of seawater, which will facilitate exploration of genetic data and life story of Coilia nasus.