Opsariichthys bidens belongs to the family Cyprinidae and is a small freshwater economic fish widely distributed in China. In recent years, the natural resources of O. bidens have been drastically reduced due to overfishing and the destruction of the water environment. The in vitro culture and long-term preservation of germ stem cells are the key technologies to keep genetic resources from degeneration. However, except for the establishment of the first long-term cultured medaka spermatogonia cell line (SSC) capable of producing sperm in vitro in 2004, no other long-term cultured SSC line has been found in other fish species. In this study, we successfully established another long-term-cultured spermatogonial stem cell line from Opsariichthys bidens (ObSSC). After more than 2 years of culture, ObSSC had a diploid karyotype and stable growth, with the typical gene expression patterns of SSC. Under in vitro culture, ObSSC could be induced to differentiate into sperm and other different types of somatic cells. In vivo, ObSSC could differentiate into different cells of three germ layers upon being transplanted into zebrafish embryos. Our research helps to explore the potential and regulation mechanism of fish SSC differentiation and spermatogenesis in vitro, provides a new way for solving the problem of fish genetic resource degradation and lays a foundation for further research on fish germ cell transplantation.

The hook snout carp Opsariichthys bidens is an important farmed fish in East Asia that shows sexual dimorphism in growth, with males growing faster and larger than females. To understand these complex traits and improve molecular breeding, chromosome-level genome assembly of male O. bidens was performed using Illumina, Nanopore, and Hi-C sequencing. The 992.9 Mb genome sequences with a contig N50 of 5.2 Mb were anchored to 38 chromosomes corresponding to male karyotypes. Of 30,922 functionally annotated genes, 97.5% of BUSCO genes were completely detected. Genome evolution analysis showed that the expanded and contracted gene families in the male O. bidens genome were enriched in 76 KEGG pathways, and 78 expanded genes were involved in the GnRH signaling pathway that regulates the synthesis and secretion of luteinizing hormone and glycoprotein hormones, further acting on male growth by inducing growth hormone. Compared to the released female O. bidens genome, the number of annotated genes in males was much higher (23,992). The male chromosome LG06 exhibited over 97% identity with the female GH14/GH38. Male-specific genes were identified for LG06, where structural variation, including deletions and insertions, occurred at a lower rate, suggesting a centric fusion of acrocentric chromosomes GH14 and GH38. The genome-synteny analysis uncovered significant inter-chromosome conservation between male O. bidens and grass carp, the former originating from ancestral chromosome breakage to increase the chromosome number. Our results provide a valuable genetic resource for studying the regulation of sexual dimorphism, sex-determining mechanisms, and molecular-guided breeding of O. bidens.

PCR-based DNA amplification has been one of the major methods in aquaculture research for decades, although its use outside the modern laboratory environment is limited due to the relatively complex methods and high costs. To this end, we investigated a swabbing and disc protocol for the collection of DNA samples from fish which could extract DNA from fish skin mucus by a non-invasion technique costing only $0.02 (USD) and requiring less than 30 seconds. The disc method that we chose could use the cheap filter paper to extract DNA from above 10⁴ crucian carp blood cells, which is comparable to the commercial kit. By using skin mucus swabbing and the disc method, we can obtain amplification-ready DNA from mucus to distinguish different species from our smallest fish (medaka, ~2.5 cm and crucian carp, ~7 cm) to our biggest fish (tilapia, ~15 cm). Furthermore, the viral pathogen Carassius auratus herpesvirus (CaHV) of crucian carp was detected using our method, which would make performing molecular diagnostic assays achievable in limited-resource settings including aquafarms and aqua stores outside the laboratory environment.