Elongate loach (Leptobotia elongata) is endemic to middle and upper reaches of the Yangtze River in China. Due to overfishing and habitat destruction, this loach has become an endangered species. So far, lack of reliable genetic information and molecular markers has hindered the conservation and utilization of elongate loach resources. Therefore, we here performed an Illumina sequencing and de novo transcriptome assembly in elongate loach, and then developed polymorphic simple sequence repeat markers (SSRs). After assembly, 51,185 unigenes were obtained, with an average length of 1,496 bp. A total of 23,901 expressed sequence tag-simple sequence repeats (EST-SSRs) were identified, distributing in 14,422 unigenes, with a distribution frequency of 28.18%. Out of 16,885 designed EST-SSR primers, 150 primers (3 or 4 base repetition-dominated) were synthesized for polymorphic EST-SSR development. Then, 52 polymorphic EST-SSRs were identified, with polymorphism information contents (PIC) ranging from 0.03 to 0.88 (average 0.54). In conclusion, this was the first report of transcriptome sequencing of elongate loach. Meanwhile, we developed a set of polymorphic EST-SSRs for the loach. This study will provide an important basis, namely genetic information and polymorphic SSRs, for further population genetics and breeding studies of this endangered and economic loach in China.

As a new freshwater aquaculture product, triploid loaches (Misgurnus anguillicaudatus) are characterized by fast growth, high-quality meat, high edibility, high resistance to disease, and sterility. In this study, a natural tetraploid loach (4n = 100) (♀) was crossed with a diploid loach (2n = 50) (♂), thus creating the hybrid triploid loach (3n = 75). The histological observations of triploid offspring and diploid controls at 4 days post-hatching (dph), 15dph, 22dph, and 50dph showed that most of the hybrid triploid loaches were abortive in the early gonad differentiation process. To explore its fertility mechanism, through transcriptome analyses of triploid offspring and diploid controls at four periods, 10 differentially expressed genes related to the early fertility mechanism were identified: amh, hormad1, rec8, h2b, plvap, zp3, h2a, nrb0b1, ddx4, and esr2. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs, two pathways were identified that are closely related to the early fertility mechanism at 50dph: the estrogen signaling pathway and steroid biosynthesis. The findings laid a foundation for further exploration of their molecular inhibition mechanism in hybrid triploid loaches.

Ammonia nitrogen is highly toxic to fish, and it can easily cause fish poisoning or even high mortality. So far, many studies have been conducted on the damages to fish under ammonia nitrogen stress. However, there are few studies of ammonia tolerance improvement in fish. In this study, the effects of ammonia nitrogen exposure on apoptosis, endoplasmic reticulum (ER) stress, and immune cells in loach Misgurnus anguillicaudatus were investigated. Loaches (60 days post fertilization) were exposed to different concentrations of NH4Cl, and their survival rates were examined every six hours. The results showed that high-concentration and long-time NH4Cl exposure (20 mM+18 h; 15 mM+36 h) induced the apoptosis and the gill tissue damages, finally causing a decline in survival. chop plays an important role in ER stress-induced apoptosis, and thus we constructed a model of chop-depleted loach by using CRISPR/Cas9 technology to investigate its response to ammonia nitrogen stress. The results showed that ammonia nitrogen stress down-regulated the expressions of apoptosis-related genes in chop+/- loach gills, while wildtype (WT) exhibited an opposite gene expression regulation pattern, suggesting that the depletion of chop suppressed apoptosis level. In addition, chop+/- loach showed a larger number of immunity-related cells and higher survival rate than WT under the NH4Cl exposure, indicating that the inhibition of chop function strengthened the innate immune barrier in general, thus increasing survival. Our findings provide the theoretical basis for developing high ammonia nitrogen-tolerant germplasm with aquaculture potential.

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The function of borealin proteins has been widely reported in the cell division of animals. Nonetheless, there is little research about their only known paralogue (cell division cycle associated 9, cdca9). In this study, cdca9 was investigated in loach (Misgurnus anguillicaudatus) for the first time. cdca9 was highly expressed in the embryo before the gastrula stage, and it was predominantly expressed in the ovary, especially in the oocytes of stage II. In conclusion, this study reveals a potential function of cdca9 in the early embryogenesis and ovarian development of fish.

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