Anguilliformes species not only possess distinctive appearance (such as body elongation and absence of pectoral and/or pelvic fins), but also display diversity in chromosome number, supporting them as a suitable model for studying karyotype evolution and related molecular mechanisms of evolutionary body patterns. However, the ancestral chromosomes and evolutionary chromosomal reorganization in various eels have not been reported yet. The most regulatory or related genes of their distinctive appearance are still unknown. Here, we predicted an eel-based ancestral chromosome karyotype for the first time, and revealed multiple chromosomal fusion and fission events that reduced the ancestral chromosome number from haploid n = 21 to the commonly extant n = 19 within the Anguilla lineage. Moreover, we carried out a genome-wide comparison of two significant gene families including homeobox (Hox) and T-box (tbx), revealing genomic loss of some Hox genes (such as HoxB9β and HoxD13α) and variation of certain tbx gene (i.e., tbx5) may be responsible for the evolutionary development of pectoral fins. Interestingly, loss of certain secretory calcium-binding phosphoprotein (SCPP) genes was identified in various eel genomes, which possibly contribute to the common reduction of scales. Overall, our current findings provide new insights into evolutionary karyotype and body pattern evolution across diverse Anguilliformes species.

DNA methylation, also known as 5-methylcytosine, is an epigenetic modification that has crucial functions in multiple important biological processes in fish, such as gonadal development. The cellular DNA methylation level is tightly regulated by DNA methyltransferases (Dnmt). However, detailed investigations of this family in fish are very scarce. In this study, our results confirmed that teleost genomes contain 4 to 16 Dnmt genes, with diversity likely resulting from a combination of whole-genome duplication (WGD), tandem duplication, and gene loss. Differences were observed in tissue distribution, transcription abundance, and protein structure of Dnmt duplicates, supporting their subfunctionalization or neofunctionalization after duplication. Interestingly, we found that fish Dnmt3b duplicates likely have acquired the functions of mammalian Dnmt3l, which may compensate for the absence of fish Dnmt3l. Furthermore, transcriptome analysis and qPCR results indicated that DNA methyltransferase genes (Dnmt1, Dnmt3aa, Dnmt3ab, Dnmt3ba, and Dnmt3bb.1) possibly play important roles in the natural sex change of protandrous hermaphrodite blackhead seabream (Acanthopagrus schlegelii) and inferred that global remodeling of gonadal DNA methylation, regulated by DNA methyltransferase genes, was closely associated with sex change in sequentially hermaphroditic fishes. Overall, our results may help provide a better understanding of the evolution and function of DNA methyltransferases in fish.

A remarkable life cycle of the protandrous blackhead seabream (Acanthopagrus schlegelii), initiating as a male during the first two years and then naturally transforming to a female since the third year, makes this fish a valuable model for studying molecular mechanisms of sex change. Here, we constructed a gap-free telomere-to-telomere (T2T) genome assembly for a male blackhead seabream, by integration of PacBio HiFi, Ultra-long ONT and Hi-C sequencing techniques. With 97.87% of the entire sequences anchored into 24 chromosomes, this haplotypic genome assembly spans 714.98 Mb. In terms of correctness (quality value QV: 52.95) and completeness (BUSCO score: 99.9%), this chromosome-scale assembly is indeed of high quality. It has been annotated with 24,581 protein-coding genes, and predicted with low percentage (30.95%) of repetitive sequences. As the first reference T2T-level genome assembly of various protandrous fishes, it provides a valuable genetic resource for expansion of fish genomics database. It will also allow for in-depth genomic comparisons among diverse hermaphrodite vertebrates, as well as offer fundamental genome data to support extensive research on blackhead seabream.

Extrachromosomal circular DNAs (eccDNAs) play a significant role in regulating various biological processes, including abnormal muscle development. The molecular functions and impact of eccDNAs in the muscle development of fish are poorly understood. To investigate the potential roles of eccDNAs in the muscle development of fish, we analyzed and compared the expression profile of muscle eccDNAs of crisp grass carp, fed a faba bean meal-based diet, and ordinary grass carp, fed a practical diet. Using the Circle-seq strategy, we found the eccDNA abundance in crisp grass carp (211,920 eccDNAs) was significantly higher than that in ordinary grass carp (25,857 eccDNAs), suggesting that the faba bean diet likely independently influences eccDNA production. Compared to ordinary grass carp, crisp grass carp exhibited 10,565 upregulated and 129 downregulated eccDNAs, indicating eccDNAs were possibly associated with the muscle development of grass carp. GO and KEGG enrichment analyses indicated that the upregulated eccDNAs were related to muscle fiber development, cellular structure, and cell junctions. Based on our results, we speculated that the overexpression of genes involved in muscle fiber, calcium metabolism, and collagen driven by eccDNAs likely contributes to the observed increase in muscle fiber density, calcium levels, and collagen content in crisp grass carp, thereby enhancing muscle hardness. Notably, eccDNAs were identified as potential innate immunostimulants capable of eliciting immune responses in fish. In summary, our findings demonstrate that eccDNAs are aberrantly expressed in the muscles of fish fed a faba bean diet, offering novel insights into the molecular mechanisms underlying muscle hardening in fish.

Slimming grass carp is a commercial variety with good body form and meat quality, which is cultured by starving common grass carp in a clean flowing water environment. Compared to common grass carp, slimming grass carp has a far higher economic value. Until now, no molecular study has concentrated on the regulation mechanism of the muscle characteristics of slimming grass carp. This study first reported the gene expression profile of the muscle characteristics of slimming grass carp based on the level of extrachromosomal circular DNAs (eccDNAs). EccDNAs are double-stranded circular DNAs derived from genomic DNAs and play crucial roles in the functional regulation of a wide range of biological processes, none of which have been shown to occur in fish. Here, muscle eccDNAs from slimming grass carp and common grass carp were both generally sequenced, and the information, as well as the expression profile of eccDNAs, were compared and analysed. The findings reveal that 82,238 and 25,857 eccDNAs were detected from slimming grass carp and common grass carp, respectively. The length distribution of eccDNAs was in the range of 1~1000 bp, with two peaks at about 200 bp and 400 bp. When the expression profiles of eccDNAs between slimming grass carp and common grass carp were compared, 3523 up-regulated and 175 down-regulated eccDNAs were found. Enrichment analysis showed that these eccDNA genes were correlated with cellular structure and response, cell immunology, enzyme activity, etc. Certain differentially expressed eccDNAs involved in muscle characteristics were detected, which include myosin heavy chain, myosin light chain, muscle segment homeobox C, calsequestrin, calmodulin, etc., among which the majority of genes were linked to muscle structure and contraction. This indicates that during the process of cultivating from common grass carp to slimming grass carp, the treatment primarily affected muscle structure and contraction, making the meat quality of slimming grass carp different from that of common grass carp. This result provides molecular evidence and new insights by which to elucidate the regulating mechanism of muscle phenotypic characterisation in slimming grass carp and other fish.

Due to potentially hostile behaviors and elusive habitats, moray eels (Muraenidae) as one group of apex predators in coral reefs all across the globe have not been well investigated. Here, we constructed a chromosome-level genome assembly for the representative Reeve’s moray eel ( Gymnothorax reevesii ). This haplotype genome assembly is 2.17 Gb in length, and 97.87% of the sequences are anchored into 21 chromosomes. It contains 56.34% repetitive sequences and 23,812 protein-coding genes, of which 96.77% are functionally annotated. This sequenced marine species in Anguilliformes makes a good complement to the genetic resource of eel genomes. It not only provides a genetic resource for in-depth studies of the Reeve’s moray eel, but also enables deep-going genomic comparisons among various eels.

Toll-like receptors 5 (TLR5), a member of the toll-like receptors (TLRs) family, is a class of pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs). It responds to vertebrate recognition of bacterial flagellin and participates in innate immune responses. However, genome-wide identification and characterization of TLR5 in fishes have not been investigated. Here, three TLR5M isotypes ( TLR5Ma , TLR5Mb1 , and TLR5Mb2 ) and a TLR5S are all extracted from fish genomes on the basis of phylogenetic and synteny analyses. We confirmed that the non-teleost fishes have one TLR5M gene, as well as additional TLR5 genes ( TLR5M and TLR5S ) in teleost fishes. In addition, some special teleost fishes possess two to three TLR5 genes, which have undergone the fourth whole-genome duplication (WGD). According to our results, we inferred that the diversity of TLR5 genes in fishes seems to be the result of combinations of WGD and gene loss. Furthermore, TLR5 isoforms displayed differences at the flagellin interaction sites and viral binding sites, and showed lineage-specific, which indicated that TLR5 duplicates may generate functional divergence. Bacterial experiments also supported the idea that CiTLR5Ma and CiTLR5Mb are subfunctionalized to sense bacterial flagellin. In summary, our present comparative genomic survey will benefit for further functional investigations of TLR5 genes in fish.

The giant freshwater prawn, Macrobrachium rosenbergii, is a prawn that has economic significance throughout the world. It exhibits sex-related growth dimorphism, whereby the males grow significantly more rapidly than the females. Therefore, a study on the molecular regulatory mechanism, which underlies the sexual differentiation of M. rosenbergii, is of both scientific and commercial importance. However, a scarcity of genomic and transcriptomic resources severely limits our knowledge of the sexual differentiation mechanisms in M. rosenbergii. Here, transcriptome sequencing of several gonadic samples of males and females in M. rosenbergii was performed to investigate the molecular basis underlying gonadal development. Our results showed that 2149 unigenes presented as differentially expressed genes (DEGs) in the ovaries of females compared to the testes of males, which contained 484 down-regulated and 1665 up-regulated genes. Enrichment analysis of DEGs revealed many of these genes to be related to sexual differentiation and gonadal development. From our transcriptome analyses, and as confirmed by quantitative real-time PCR, male-related genes (Mrr, MRPINK, IR, IAGBP, TESK1, and dsx) in the testes were significantly up-regulated, and female-related genes (ERR, Sxl3, cyclinB, Dmrt99B, PPP2A, and ADCY9) in the ovaries were also significantly up-regulated. This indicates the potential role these genes play in the gonadal development of M. rosenbergii. Furthermore, multiple signal transduction pathways relating to gonadal maturation and spermatogenesis, including MAPK, were identified herein. Our data also supports previous ideas that IAG and IAGBP-IR signaling schemes could help in the regulation of testis’ development in M. rosenbergii and the ERR gene could regulate ovarian development by affecting the expression of cyclinB, PPP2A, and ADCY9. The data from this study provides incredibly usefully genomic resources for future research on the sexual differentiation and practical aquaculture of M. rosenbergii.

The 14-3-3 family genes are highly conserved regulatory factors in eukaryotes with involvement in multiple important cellular processes. However, detailed investigations of this family in fishes are very limited. Here, a comparative genomic and transcriptomic survey were performed to investigate the 14-3-3 family in fishes. We confirmed that the numbers of 14-3-3 genes ranged from 5 to 7 in non-teleost fishes, as well as additional 14-3-3 genes (9 to 11) in teleost fishes. In addition, some special teleost fishes possess 17 to 25 14-3-3s, which undergone the fourth whole-genome duplication (WGD). We also found that six pairs of fish 14-3-3 genes were clustered with mammalian ε, γ, ς, η, τand β isotypes, respectively, while σ was absent with a potential specificity within mammals, on the basis of their phylogenetic and synteny analyses. According to our results, we inferred that the diversity of 14-3-3 genes in fishes seems to be generated from a combination of WGD and gene loss. Comparative transcriptomic analysis revealed that there are differences in tissue distribution, and we speculated that 14-3-3 genes may contribute to terrestrial adaptations in mudskippers. In addition, protein sequence alignments of 14-3-3s supported their differential roles in fishes. In summary, our present comparative genomic and transcriptomic survey will benefit for further functional investigations of these fish genes.

Although there are various Conus species with publicly available transcriptome and proteome data, no genome assembly has been reported yet. Here, using Chinese tubular cone snail ( C. betulinus ) as a representative, we sequenced and assembled the first Conus genome with original identification of 133 genome-widely distributed conopeptide genes. After integration of our genomics, transcriptomics, and peptidomics data in the same species, we established a primary genetic central dogma of diverse conopeptides, assuming a rough number ratio of ~1:1:1:10s for the total genes: transcripts: proteins: post-translationally modified peptides. This ratio may be special for this worm-hunting Conus species, due to the high diversity of various Conus genomes and the big number ranges of conopeptide genes, transcripts, and peptides in previous reports of diverse Conus species. Only a fraction (45.9%) of the identified conotopeptide genes from our achieved genome assembly are transcribed with transcriptomic evidence, and few genes individually correspond to multiple transcripts possibly due to intraspecies or mutation-based variances. Variable peptide processing at the proteomic level, generating a big diversity of venom conopeptides with alternative cleavage sites, post-translational modifications, and N-/C-terminal truncations, may explain how the 133 genes and ~123 transcripts can generate thousands of conopeptides in the venom of individual C. betulinus . We also predicted many conopeptides with high stereostructural similarities to the putative analgesic ω-MVIIA, addiction therapy AuIB and insecticide ImI, suggesting that our current genome assembly for C. betulinus is a valuable genetic resource for high-throughput prediction and development of potential pharmaceuticals.

Mitochondrial genomes (mitogenomes) are an excellent source of information for phylogenetic and evolutionary studies, but their application in marine invertebrates is limited. In the present study, we utilized mitogenomes to elucidate the phylogeny and environmental adaptation in deep-sea mussels (Mytilidae: Bathymodiolinae). We sequenced and assembled seven bathymodioline mitogenomes. A phylogenetic analysis integrating the seven newly assembled and six previously reported bathymodioline mitogenomes revealed that these bathymodiolines are divided into three well-supported clades represented by five Gigantidas species, six Bathymodiolus species, and two "Bathymodiolus" species, respectively. A Common interval Rearrangement Explorer (CREx) analysis revealed a gene order rearrangement in bathymodiolines that is distinct from that in other shallow-water mytilids. The CREx analysis also suggested that reversal, transposition, and tandem duplications with subsequent random gene loss (TDRL) may have been responsible for the evolution of mitochondrial gene orders in bathymodiolines. Moreover, a comparison of the mitogenomes of shallow-water and deep-sea mussels revealed that the latter lineage has experienced relaxed purifying selection, but 16 residues of the atp6, nad4, nad2, cob, nad5, and cox2 genes have underwent positive selection. Overall, this study provides new insights into the phylogenetic relationships and mitogenomic adaptations of deep-sea mussels

Giant freshwater prawns (Macrobrachium rosenbergii) are commonly found throughout the world. The size of the male giant freshwater prawn is much larger than that of the female. Therefore, understanding the molecular mechanism that underlies the sexual differentiation of M. rosenbergii is of both commercial and scientific importance. Insulin-like androgenic gland hormone (IAG) plays a key role in the differentiation of sex in M. rosenbergii. Although IAG has been investigated, the regulatory relationship between IAG and its binding protein partner, the insulin-like androgenic gland hormone-binding protein (IAGBP), has not been studied in M. rosenbergii. Here, we cloned and characterized the IAGBP from M. rosenbergii (Mr-IAGBP) for the very first time. Transcriptomic analysis showed that Mr-IAGBP mRNA was detected in a wide array of tissues with the highest expression found in the androgenic gland. The importance of IAG in male development was further demonstrated by an increase in IAG transcripts during the development of the androgenic gland and Mr-IAG was only highly transcribed in the androgenic gland of M. rosenbergii. Interestingly, we found that the Mr-IAG gene expression started during the 20th-day larva after hatching stage (LH20), followed (20th-day post-larval stage, PL20) by a gradual elevation of Mr-IAGBP levels. The levels of both genes peaked at the adult stage. The relationship between Mr-IAGBP and Mr-IAG was further analyzed using RNA interference. The injection of Mr-IAGBP double-stranded RNA (dsRNA) significantly reduced the transcription of Mr-IAG, while the amount of Mr-IAGBP mRNA and the translation of IAGBP protein was significantly reduced by the injection of Mr-IAG dsRNA. These results revealed that IAGBP is involved in IAG signaling. Furthermore, our data supports the hypothesis that (IAG and IAGBP)-IAG receptor signaling schemes exist in M. rosenbergii. Our results will provide important information for the further study of determining the sex of M. rosenbergii.

Intermuscular bones (IBs) are only found in the muscles of fish. Bone morphogenetic protein 2 (bmp2) is considered to be the most active single osteogenesis factor. It promotes cell proliferation and differentiation during bone repair, as well as inducing the formation of bones and cartilages in vivo. However, detailed investigations of this family in fish are incredibly limited. Here, we have used a variety of published and unpublished bmp2 sequences for teleosts and cartilage fish in order to explore and expand our understanding of bmp2 genes in fish. Our results confirmed that teleost genomes contain two or more bmp2 genes, and the diversity of bmp2 genes in vertebrates appears to be as a result of a combination of whole genome duplication (WGD) and gene loss. Differences were also observed in tissue distribution and relative transcription abundance of the bmp2s through a transcriptomic analysis. Our data also indicated that bmp2b may play an important role in the formation of IBs in teleosts. In addition, protein sequence alignments and 3D structural predictions of bmp2a and bmp2b supported their similar roles in fishes. To summarize, our existing work provided novel insights into the bmp2 family genes in fishes through a mixture of comparative genomic and transcriptomic analysis.

Protandrous black porgy (Acanthopagrus schlegelii) is a popular and valuable commercial marine fish in China and East Asian countries. Controlling and managing its breeding has been an imperative step towards obtaining a sustainable supply of this fish in aquaculture production systems. Therefore, study on the molecular mechanisms of sex change in black porgy has both scientific and commercial importance. Previously, we identified some candidate genes related to sex determination and differentiation from a high-quality genome assembly of the black porgy. In the present study, transcriptome sequencing of developmental gonads (including testis, ovotestis and ovary) of black porgy was performed to further investigate the sex-change mechanisms. Our results showed that the highly expressed male-related genes (dmrt1, piwi1, piwi2, sox9, sox30 and amh) at the male phase were significantly down-regulated to a substantial degree at the intersexual stage, and the female-related genes (jnk1, vasa, wnt4, figla and foxl2) were distinctly up-regulated when the fish grows into a female adult, suggesting the potential roles of these genes in sex change of the black porgy. These data also support a previous hypothesis that the femaleness will be switched on when the testis is entering the degenerated stage through the diminished dmrt1 expression. Our transcriptome data provide a very useful genomic resource for future studies on sex change and practical aquaculture in the black porgy.

As a living fossil, the endangered Chinese sturgeon (Acipenser sinensis) has been considered a national treasure in China. Here, the famous Gezhouba Dam and Three Gorges Dam on the Yangtze River were built in 1988 and 2006, for economic purposes. The natural population of Chinese sturgeon has declined since then, as these dams block its migratory route to the original spawning grounds in the middle reaches of the Yangtze River. In 2013 and 2014, there was an absence of spawning where it typically happened near the Gezhouba Dam. Nevertheless, from April to June in 2015, over 1,000 larvae with different body lengths (10–35 cm) were detected along the Shanghai Yangtze Estuary; but only little is currently known about the population genetic structure of the Chinese sturgeon. Herein, we inferred population genetic parameters from 462 available Chinese sturgeon specimens based on a 421-bp fragment of the mitochondrial DNA (mtDNA) D-loop region and 1,481,620 SNPs (single-nucleotide polymorphisms) generated by restriction site-associated DNA sequencing (RAD-seq). For the D-loop dataset, 15 haplotypes were determined. Randomly picked 23 individuals, representing the 15 D-loop haplotype groups, were subsequently used for further RAD-seq validation. The average nucleotide diversity calculated from the mtDNA and RAD datasets was 0.0086 and 0.000478, respectively. The overall effective female population size was calculated to be 1,255 to 2,607, and the long-term effective population size was estimated to range from 11,950 to 119,500. We observed that the genetic variability and the effective female population size of the current population in the Yangtze River are severely low, which are similar to the data reported over 10 years ago. The deduced relatively small effective population of female fish, limiting the genetic connectivity among Chinese sturgeon, should be considered a serious threat to this endangered species.