Objectives: Swamp eel is one model species for sexual reversion and an aquaculture fish in China. One local strain with deep yellow and big spots of Monopterus albus has been selected for consecutive selective breeding. The objectives of this study were characterizing the Simple Sequence Repeats (SSRs) of M. albus in the assembled genome obtained recently, and developing polymorphic SSRs for future breeding programs. Methods: The genome wide SSRs were mined by using MISA software, and their types and genomic distribution patterns were investigated. Based on the available flanking sequences, primer pairs were batched developed, and Polymorphic SSRs were identified by using Polymorphic SSR Retrieval tool. The obtained polymorphic SSRs were validated by using e-PCR and capillary electrophoresis, then they were used to investigate genetic diversity of one breeding population. Results: A total of 364,802 SSRs were identified in assembled M. albus genome. The total length, density and frequency of SSRs were 8,204,641 bp, 10,259 bp/Mb, and 456.16 loci/Mb, respectively. Mononucleotide repeats were predominant among SSRs (33.33%), and AC and AAT repeats were the most abundant di- and tri-nucleotide repeats motifs. A total of 287,189 primer pairs were designed, and a high-density physical map was constructed (359.11 markers per Mb). A total of 871 polymorphic SSRs were identified, and 38 SSRs of 101 randomly selected ones were validated by using e-PCR and capillary electrophoresis. Using these 38 polymorphic SSRs, 201 alleles were detected and genetic diversity level (Na, PIC, H_O, and H_e) was evaluated. Conclusions: The genome-wide SSRs and newly developed SSR markers will provide a useful tool for genetic mapping, diversity analysis studies in swamp eel in the future. The high level of genetic diversity (Na = 5.29, PIC = 0.5068, H_O = 0.4665, H_e = 0.5525) but excess of homozygotes ( F_IS = 0.155) in one breeding population provide baseline information for future breeding program.

As a nucleolar complex for small-subunit (SSU) ribosomal RNA processing, SSU processome has been extensively studied mainly in Saccharomyces cerevisiae but not in diverse organisms, leaving open the question of whether it is a ubiquitous mechanism across eukaryotes and how it evolved in the course of the evolution of eukaryotes. Genome-wide survey and identification of SSU processome components showed that the majority of all 77 yeast SSU processome proteins possess homologs in almost all of the main eukaryotic lineages, and 14 of them have homologs in archaea but few in bacteria, suggesting that the complex is ubiquitous in eukaryotes, and its evolutionary history began with abundant protein homologs being present in archaea and then a fairly complete form of the complex emerged in the last eukaryotic common ancestor (LECA). Phylogenetic analysis indicated that ancient gene duplication and functional divergence of the protein components of the complex occurred frequently during the evolutionary origin of the LECA from prokaryotes. We found that such duplications not only increased the complex’s components but also produced some new functional proteins involved in other nucleolar functions, such as ribosome biogenesis and even some nonnucleolar (but nuclear) proteins participating in pre-mRNA splicing, implying the evolutionary emergence of the subnuclear compartment—the nucleolus—has occurred in the LECA. Therefore, the LECA harbored not only complicated SSU processomes but also a nucleolus. Our analysis also revealed that gene duplication, innovation, and loss, caused further divergence of the complex during the divergence of eukaryotes.

In fish, sperm motility activation is one of the most essential procedures for fertilization. Previous studies have mainly focused on the external environmental effects and intracellular signals in sperm activation; however, little is known about the metabolic process of sperm motility activation in fish. In the present study, using ricefield eel (Monopterus albus) sperm as a model, metabonomics was used to analyze the metabolic mechanism of the sperm motility activation in fish. Firstly, 529 metabolites were identified in the sperm of ricefield eel, which were clustered into the organic acids, amino acids, nucleotides, benzene, and carbohydrates, respectively. Among them, the most abundant metabolites in sperm were L-phenylalanine, DL-leucine, L-leucine, lysolecithin choline 18:0, L-tryptophan, adenine, hypoxanthine, 7-Methylguanine, shikimic acid, and L-tyrosine. Secondly, compared to pre-activated sperm, the level of S-sulfo-L-cysteine and L-asparagine were both increased in the post-activated sperm. Ninety-two metabolites were decreased in the post-activated sperm, including quinic acid, acetylsalicylic acid, 7,8-dihydro L-biopterin, citric acid, glycylphenylalanine, and dihydrotachysterol (DHT). Finally, basing on the pathway analysis, we found that the changed metabolites in sperm motility activation were mainly clustered into energy metabolism and anti-oxidative stress. Fish sperm motility activation would be accompanied by the release of a large amount of energy, which might damage the genetic material of sperm. Thus, the anti-oxidative stress function is a critical process to maintain the normal physiological function of sperm.

Asian swamp eel (Monopterus albus, Zuiew 1793) is a commercially important fish due to its nutritional value in Eastern and Southeastern Asia. One local strain of M. albus distributed in the Jianghan Plain of China has been subjected to a selection breeding program because of its preferred body color and superiority of growth and fecundity. Some members of the genus Monopterus have been reclassified into other genera recently. These classifications require further phylogenetic analyses. In this study, the complete mitochondrial genomes of the breeds of M. albus were decoded using both PacBio and Illumina sequencing technologies, then phylogenetic analyses were carried out, including sampling of M. albus at five different sites and 14 species of Synbranchiformes with complete mitochondrial genomes. The total length of the mitogenome is 16,621 bp, which is one nucleotide shorter than that of four mitogenomes of M. albus sampled from four provinces in China, as well as one with an unknown sampling site. The gene content, gene order, and overall base compositions are almost identical to the five reported ones. The results of maximum likelihood (ML) and Bayesian inference analyses of the complete mitochondrial genome and 13 protein-coding genes (PCGs) were consistent. The phylogenetic trees indicated that the selecting breed formed the deepest branch in the clade of all Asian swamp eels, confirmed the phylogenetic relationships of four genera of the family Synbranchidae, also providing systematic phylogenetic relationships for the order Synbranchiformes. The divergence time analyses showed that all Asian swamp eels diverged about 0.49 million years ago (MYA) and their common ancestor split from other species about 45.96 MYA in the middle of the Miocene epoch. Altogether, the complete mitogenome of this breed of M. albus would serve as an important dataset for germplasm identification and breeding programs for this species, in addition to providing great help in identifying the phylogenetic relationships of the order Synbranchiformes.

Extrachromosomal circular DNA (eccDNA) serves as a vital regulator of gene expression and phenotypic plasticity. The Monopterus albus is a protogynous hermaphroditic fish, and the functional role of eccDNA during its female-to-male sex reversal remains poorly understood. We conducted Circle-seq analysis on gonadal tissues of super-female and male individuals and integrated our findings with previously published gonadal RNA-seq data. A total of 62,574 and 34,562 eccDNAs were, on average, identified in the two groups, and 7881 down-regulated and 2738 up-regulated differentially abundant eccDNAs were screened in male gonads. Three candidate eccDNAs, eccDNA^Dmrt1a, eccDNA^Sox9a1 and eccDNA^ZNF239, were selected for subsequent analysis. Quantitative real-time PCR was applied to detect the overall transcriptional levels of their corresponding host genes, covering transcripts derived from both genomic DNA and eccDNA. Spatiotemporal expression profiling and letrozole induction experiments revealed male-biased expression of Dmrt1a and Sox9a1, while ZNF239 was predominantly expressed in female gonads. Complete sex reversal was observed in female M. albus after 90-day letrozole treatment, accompanied by elevated Dmrt1a/Sox9a1 and reduced ZNF239 transcription. Fluorescence in situ hybridization localized these transcripts in gonadal germ cells and somatic cells across developmental stages. This study verifies the production of eccDNA during gonadal development. The results indicate that Dmrt1a and Sox9a1 may facilitate sex reversal and suggest that ZNF239 is likely involved in ovarian development. This work provides novel clues for deciphering the molecular mechanism underlying sex reversal from the eccDNA perspective.