Whole-Genome Sequencing Analyses Reveal the Evolution Mechanisms of Typical Biological Features of Decapterus maruadsi.

Decapterus maruadsi is a typical representative of small pelagic fish characterized by fast growth rate, small body size, and high fecundity. It is a high-quality marine commercial fish with high nutritional value. However, the underlying genetics and genomics research focused on D. maruadsi is not comprehensive. Herein, a high-quality chromosome-level genome of a male D. maruadsi was assembled. The assembled genome length was 716.13 Mb with contig N50 of 19.70 Mb. Notably, we successfully anchored 95.73% contig sequences into 23 chromosomes with a total length of 685.54 Mb and a scaffold N50 of 30.77 Mb. A total of 22,716 protein-coding genes, 274.90 Mb repeat sequences, and 10,060 ncRNAs were predicted, among which 22,037 (97%) genes were successfully functionally annotated. The comparative genome analysis identified 459 unique, 73 expanded, and 52 contracted gene families. Moreover, 2804 genes were identified as candidates for positive selection, of which some that were related to the growth and development of bone, muscle, cardioid, and ovaries, such as some members of the TGF-ß superfamily, were likely involved in the evolution of typical biological features in D. maruadsi. The study provides an accurate and complete chromosome-level reference genome for further genetic conservation, genomic-assisted breeding, and adaptive evolution research for D. maruadsi.

Liver transcriptome analysis reveal the metabolic and apoptotic responses of Trachinotus ovatus under acute cold stress.

Trachinotus ovatus is an economically important fish and has been recommended as a high-quality aquaculture fish breed for the high-quality development of sea ranches in the South China Sea. However, T. ovatus shows intolerance to low temperature, greatly limiting the extension of farming scale, reducing production efficiency in winter, and increasing farming risks. In this study, liver transcriptome analysis was investigated in T. ovatus under acute low temperature conditions (20 and 15 °C) using RNA sequencing (RNA-Seq) technology. Inter-groups differential expression analysis and trend analysis screened 1219 DEGs and four significant profiles (profiles 0, 3, 4, and 7), respectively. GO enrichment analysis showed that these DEGs were mainly related to metabolic process and cell growth and death process. KEGG enrichment analysis found that DEGs were mainly associated with lipid metabolism, carbohydrate metabolism, and cell growth and death, such as gluconeogenesis, glycolysis, fatty acid oxidation, cholesterol biosynthesis, p53 signaling pathway, cell cycle arrest, and apoptotic cell death. Moreover, protein-protein interaction networks identified two hub genes (FOS and JUNB) and some important genes related to metabolic process and cell growth and death process, that corresponding to enrichment analysis. Overall, gluconeogenesis, lipid mobilization, and fatty acid oxidation in metabolic process and cell cycle arrest and apoptotic cell death in cell growth and death process were enhanced, while glycolysis, liver glycogen synthesis and cholesterol biosynthesis in metabolic process were inhibited. The enhancement or attenuatment of metabolic process and cell growth and death process is conducive to maintain energy balance, normal fluidity of cell membrane, normal physiological functions of liver cell, enhancing the tolerance of T. ovatus to cold stress. These results suggested that metabolic process and cell growth and death process play important roles in response to acute cold stress in the liver of T. ovatus. Gene expreesion level analysis showed that acute cold stress at 15 °C was identified as a critical temperature point for T. ovatus in term of cellular metabolism alteration and apoptosis inducement, and rewarming intervention should be timely implemented above 15 °C. Our study can provide theoretical support for breeding cold-tolerant cultivars of T. ovatus, which is contributed to high-quality productions fish production.

Whole-Genome Sequencing Analyses Reveal the Whip-like Tail Formation, Innate Immune Evolution, and DNA Repair Mechanisms of Eupleurogrammus muticus.

Smallhead hairtail (Eupleurogrammus muticus) is an important marine economic fish distributed along the northern Indian Ocean and the northwest Pacific coast; however, little is known about the mechanism of its genetic evolution. This study generated the first genome assembly of E. muticus at the chromosomal level using a combination of PacBio SMRT, Illumina Nova-Seq, and Hi-C technologies. The final assembled genome size was 709.27 Mb, with a contig N50 of 25.07 Mb, GC content of 40.81%, heterozygosity rate of 1.18%, and repetitive sequence rate of 35.43%. E. muticus genome contained 21,949 protein-coding genes (97.92% of the genes were functionally annotated) and 24 chromosomes. There were 143 expansion gene families, 708 contraction gene families, and 4888 positively selected genes in the genome. Based on the comparative genomic analyses, we screened several candidate genes and pathways related to whip-like tail formation, innate immunity, and DNA repair in E. muticus. These findings preliminarily reveal some molecular evolutionary mechanisms of E. muticus at the genomic level and provide important reference genomic data for the genetic studies of other trichiurids.

Integrated Transcriptomics and Metabolomics Reveal Changes in Cell Homeostasis and Energy Metabolism in Trachinotus ovatus in Response to Acute Hypoxic Stress.

Trachinotus ovatus is an economically important mariculture fish, and hypoxia has become a critical threat to this hypoxia-sensitive species. However, the molecular adaptation mechanism of T. ovatus liver to hypoxia remains unclear. In this study, we investigated the effects of acute hypoxic stress (1.5 ± 0.1 mg·L-1 for 6 h) and re-oxygenation (5.8 ± 0.3 mg·L-1 for 12 h) in T. ovatus liver at both the transcriptomic and metabolic levels to elucidate hypoxia adaptation mechanism. Integrated transcriptomics and metabolomics analyses identified 36 genes and seven metabolites as key molecules that were highly related to signal transduction, cell growth and death, carbohydrate metabolism, amino acid metabolism, and lipid metabolism, and all played key roles in hypoxia adaptation. Of these, the hub genes FOS and JUN were pivotal hypoxia adaptation biomarkers for regulating cell growth and death. During hypoxia, up-regulation of GADD45B and CDKN1A genes induced cell cycle arrest. Enhancing intrinsic and extrinsic pathways in combination with glutathione metabolism triggered apoptosis; meanwhile, anti-apoptosis mechanism was activated after hypoxia. Expression of genes related to glycolysis, gluconeogenesis, amino acid metabolism, fat mobilization, and fatty acid biosynthesis were up-regulated after acute hypoxic stress, promoting energy supply. After re-oxygenation for 12 h, continuous apoptosis favored cellular function and tissue repair. Shifting from anaerobic metabolism (glycolysis) during hypoxia to aerobic metabolism (fatty acid ß-oxidation and TCA cycle) after re-oxygenation was an important energy metabolism adaptation mechanism. Hypoxia 6 h was a critical period for metabolism alteration and cellular homeostasis, and re-oxygenation intervention should be implemented in a timely way. This study thoroughly examined the molecular response mechanism of T. ovatus under acute hypoxic stress, which contributes to the molecular breeding of hypoxia-tolerant cultivars.

Transcriptomic Response of the Liver Tissue in Trachinotus ovatus to Acute Heat Stress.

Trachinotus ovatus is a major economically important cultured marine fish in the South China Sea. However, extreme weather and increased culture density result in uncontrollable problems, such as increases in water temperature and a decline in dissolved oxygen (DO), hindering the high-quality development of aquaculture. In this study, liver transcriptional profiles of T. ovatus were investigated under acute high-temperature stress (31 °C and 34 °C) and normal water temperature (27 °C) using RNA sequencing (RNA-Seq) technology. Differential expression analysis and STEM analysis showed that 1347 differentially expressed genes (DEGs) and four significant profiles (profiles 0, 3, 4, and 7) were screened, respectively. Of these DEGs, some genes involved in heat shock protein (HSPs), hypoxic adaptation, and glycolysis were up-regulated, while some genes involved in the ubiquitin-proteasome system (UPS) and fatty acid metabolism were down-regulated. Our results suggest that protein dynamic balance and function, hypoxia adaptation, and energy metabolism transformation are crucial in response to acute high-temperature stress. Our findings contribute to understanding the molecular response mechanism of T. ovatus under acute heat stress, which may provide some reference for studying the molecular mechanisms of other fish in response to heat stress.

Chromosome-Level Genome Assembly Provides Insights into the Evolution of the Special Morphology and Behaviour of Lepturacanthus savala.

Savalani hairtail Lepturacanthus savala is a widely distributed fish along the Indo-Western Pacific coast, and contributes substantially to trichiurid fishery resources worldwide. In this study, the first chromosome-level genome assembly of L. savala was obtained by PacBio SMRT-Seq, Illumina HiSeq, and Hi-C technologies. The final assembled L. savala genome was 790.02 Mb with contig N50 and scaffold N50 values of 19.01 Mb and 32.77 Mb, respectively. The assembled sequences were anchored to 24 chromosomes by using Hi-C data. Combined with RNA sequencing data, 23,625 protein-coding genes were predicted, of which 96.0% were successfully annotated. In total, 67 gene family expansions and 93 gene family contractions were detected in the L. savala genome. Additionally, 1825 positively selected genes were identified. Based on a comparative genomic analysis, we screened a number of candidate genes associated with the specific morphology, behaviour-related immune system, and DNA repair mechanisms in L. savala. Our results preliminarily revealed mechanisms underlying the special morphological and behavioural characteristics of L. savala from a genomic perspective. Furthermore, this study provides valuable reference data for subsequent molecular ecology studies of L. savala and whole-genome analyses of other trichiurid fishes.

Molecular characterisation and biological activity of an antiparasitic peptide from Sciaenops ocellatus and its immune response to Cryptocaryon irritans.

Cryptocaryon irritans, a holotrichous ciliate parasitic protozoan, can trigger marine white spot disease and cause substantial economic losses in mariculture. However, methods of preventing and curing the disease have negatively affect fish, human, other organisms, and the natural environment. The antiparasitic activity of some antimicrobial peptides (AMPs) has garnered extensive attention of scholars. In this study, we identified and characterised a novel antiparasitic peptide, named So-pis, from Sciaenops ocellatus. The sequence analysis, structural features, and tissue distribution suggested that So-pis is genetically related to the piscidins family. However, So-pis showed a relatively low overall conservation compared with other known piscidins. So-pis is abound in glycine residues (22.7 %) and it has a neutral isoelectric point, weak amphipathicity, relatively long α-helix, and high hydrophobicity. These key elements are responsible for its biological activity. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicated that So-pis is a typically gill-expressed peptide. The expression of So-pis in the gill, skin, spleen, and head kidney could be regulated during C. irritans infection, thereby implicating a role of So-pis in immune defence against C. irritans. The synthetic So-pis had limited or no antimicrobial activity against bacterial and yeasts but exhibited potent antiparasitic activity against C. irritans in vitro. The activity of synthetic So-pis against erythrocytes was less potent than its antiparasitic activity against C. irritans. These results indicated that So-pis might be one of the crucial defence cytokines against C. irritans in the red drum. Cumulatively, our data suggested that So-pis might be a potential candidate for developing a novel, effective, and safe therapeutic agent against marine white spot disease.

Gene Expression Profile and Co-Expression Network of Pearl Gentian Grouper under Cold Stress by Integrating Illumina and PacBio Sequences.

Pearl gentian grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) is a fish of high commercial value in the aquaculture industry in Asia. However, this hybrid fish is not cold-tolerant, and its molecular regulation mechanism underlying cold stress remains largely elusive. This study thus investigated the liver transcriptomic responses of pearl gentian grouper by comparing the gene expression of cold stress groups (20, 15, 12, and 12 °C for 6 h) with that of control group (25 °C) using PacBio SMRT-Seq and Illumina RNA-Seq technologies. In SMRT-Seq analysis, a total of 11,033 full-length transcripts were generated and used as reference sequences for further RNA-Seq analysis. In RNA-Seq analysis, 3271 differentially expressed genes (DEGs), two low-temperature specific modules (tan and blue modules), and two significantly expressed gene sets (profiles 0 and 19) were screened by differential expression analysis, weighted gene co-expression networks analysis (WGCNA), and short time-series expression miner (STEM), respectively. The intersection of the above analyses further revealed some key genes, such as PCK, ALDOB, FBP, G6pC, CPT1A, PPARα, SOCS3, PPP1CC, CYP2J, HMGCR, CDKN1B, and GADD45Bc. These genes were significantly enriched in carbohydrate metabolism, lipid metabolism, signal transduction, and endocrine system pathways. All these pathways were linked to biological functions relevant to cold adaptation, such as energy metabolism, stress-induced cell membrane changes, and transduction of stress signals. Taken together, our study explores an overall and complex regulation network of the functional genes in the liver of pearl gentian grouper, which could benefit the species in preventing damage caused by cold stress.

Demographic history and population genetic analysis of Decapterus maruadsi from the northern South China Sea based on mitochondrial control region sequence.

Late Pleistocene climate oscillations are believed to have greatly influenced the distribution, population dynamics, and genetic variation of many marine organisms in the western Pacific. However, the impact of the late Pleistocene climate cycles on the demographic history and population genetics of pelagic fish in the northern South China Sea (SCS) remains largely unexplored. In this study, we explored the demographic history, genetic structure, and genetic diversity of Decapterus maruadsi, a typical pelagic fish, over most of its range in the northern SCS. A 828-832 bp fragment of mitochondrial control region were sequenced in 241 individuals from 11 locations. High haplotype diversity (0.905-0.980) and low nucleotide diversity (0.00269-0.00849) was detected, revealing low levels of genetic diversity. Demographic history analysis revealed a pattern of decline and subsequent rapid growth in the effective population size during deglaciation, which showed that D. maruadsi experienced recent demographic expansion after a period of low effective population size. Genetic diversity, genetic structure, and phylogenetic relationship analysis all demonstrated that no significant genetic differentiation existed among the populations, indicating that D. maruadsi was panmictic throughout the northern SCS. Periodic sea-level changes, fluctuation of the East Asian Monsoon, and Kuroshio variability were responsible for the population decline and expansion of D. maruadsi. The demographic history was the primary reason for the low levels of genetic diversity and the lack of significant genetic structure. The life history characteristics and ocean currents also had a strong correlation with the genetic homogeneity of D. maruadsi. However, the genetic structure of the population (genetic homogeneity) is inconsistent with biological characteristics (significant difference), which is an important reminder to identify and manage the D. maruadsi population carefully.

Complete mitochondrial genome of yellowback seabream, Dentex hypselosomus and phylogenetic analysis of the family Sparidae.

The complete mitogenome sequence of Dentex hypselosomus was amplified by designing 15 primer pairs. The circle genome was 16,618 bp in size and the overall base composition was 27.04% of A, 26.38% of T, 17.08% of G, and 29.50 of C, with significant anti-G bias. The complete mitogenome of D. hypselosomus encodes 37 canonical mitochondrial genes, two non-coding regions, an L-strand replication region (OL), and a control region (D-loop). The D-loop contained termination sequence domain (TAS), central conserved domains (CSB-F, CSB-E, CSB-D, CSB-C, and CSB-A), and conserved sequence blocks (CSB-1, CSB-2, and CSB-3). Phylogenetic analysis of nine sparid species well supported the phylogenetic position of D. hypselosomus and revealed the phylogenetic relationship of the family Sparidae at the level of mitochondrial genomes.

Complete mitochondrial genome and phylogenetic relationship of ornate threadfin bream, Nemipterus hexodon (Perciformes, Nemipteridae).

The complete mitochondrial genome of the ornate threadfin bream, Nemipterus hexodon, was first determined by the pairs-walking sequencing in this study. The circular mtDNA molecule was 17,115 bp in size and the overall nucleotide composition of H-stand was A (29.55%), T (27.36%), G (16.08%), and C (27.01%), with a slight bias towards A + T. The complete mitogenome encoded 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions (an origin of L-strand replication and a control region). The Bayesian tree supported the phylogenetic position of N. hexodon, which provided useful information for phylogenetic relationship in genus Nemipterus.

The second complete mitogenome of Nemipterus virgatus to dissect control region structure and phylogenetic problem of the superfamily Sparoidea (Teleostei, Perciformes).

In this study, we determined the complete mitogenome of Nemipterus virgatus of which the length was 17,073 bp, including 37 canonical mitochondrial genes and 2 non-coding regions. The control region contained termination associated sequence domain (TAS), central conserved domain (CSB-F, CSB-E, CSB-D, CSB-C, and CSB-A), conserved sequence block domain (CSB-1, CSB-2, and CSB-3), and tandem repeat sequence domain (TTD). Nine single nucleotide polymorphisms and three insertion of tandem repeat sequence (each length in 28 bp) were detected between two N. virgatus mitogenomes. The phylogenetic analysis showed that the families Nemipteridae, Sparidae, Centracanthidae, and Lethrinidae did not gather into a monophyly of superfamily Sparoidea in the neighbor-joining tree.

The second complete mitogenome of Scolopsis ciliata (Perciformes, Nemipteridae) to analyze control region structure and intraspecific variation.

In this study, we reported the complete mitogenome of Scolopsis ciliata sampled from the Beibu Gulf, South China Sea. It was 16,734 bp in length and contained 37 canonical mitochondrial genes and 2 non-coding regions. Fifty-one single nucleotide polymorphisms and five insertion and deletions were detected between two S. ciliata mitogenomes, suggesting a high level of intraspecific genetic variation in the species. The control region contained termination associated sequence domain (TAS), central conserved domain (CSB-F, CSB-E, CSB-D, CSB-C, and CSB-A), and conserved sequence block domain (CSB-1, CSB-2, and CSB-3). The phylogenetic analysis of 21 complete mitogenome sequences well supports the phylogenetic position of S. ciliata and reveals the phylogenetic relationship of the genus Scolopsis.

Identification and expression analysis of a new invertebrate lysozyme in Kuruma shrimp (Marsupenaeus japonicus).

Lysozyme is an important component of the innate immunity system against invading pathogens. An invertebrate (i-type) lysozyme from the hepatopancreas of Kuruma shrimp Marsupenaeus japonicus (Mj-ilys) was identified. The full-length cDNA of Mj-ilys was 580bp with a 429 bp open reading frame encoding a 142 amino acid polypeptide. The encoded polypeptide was predicted to have a 17 amino acid signal peptide, and a 125 amino acid mature protein with a theoretical mass of 14.099 kDa and an isoelectric point (pI) of 4.18. A Destabilase conserved domain was predicted in Mj-ilys amino acid sequences which may be stable by 10 cysteine residues forming 5 disulfide bonds. Mj-ilys may loss the muramidase and isopeptidase activities due to the lack of the key catalytic residues. Mj-ilys had high homologous of 80-82% with i-type lysozymes of penaeid shrimps. It was first grouped with other i-type lysozyme of shrimps and crabs in a phylogenetic tree predicted by the Neighbor-Joining method. Mj-ilys mRNA was expressed mainly in hepatopancreas and almost undetectable in other tissues. The mRNA expression of Mj-ilys were all found from fertilized eggs to post-larvae of 17 days (PL17), and its expression exhibited significant differences among each developmental stage. After white spot syndrome virus (WSSV) challenge (3.6 × 10(8) virions/µl), the time-dependent expression pattern of Mj-ilys in hepatopancreas and gills showed significantly different. These results indicated that Mj-ilys is potentially involved in the ontogenesis and immune defense in Kuruma shrimp.

Identification and expression analysis of a novel stylicin antimicrobial peptide from Kuruma shrimp (Marsupenaeus japonicus).

Antimicrobial peptides (AMPs) are important components of the innate immune system and function as the first line of defense against invading pathogens. In current study we identified, cloned and characterized a novel stylicin AMP from Kuruma shrimp Marsupenaeus japonicus (Mj-sty). The full-length cDNA of Mj-sty was 428 bp with an open reading frame of 315 bp that encoded 104 amino acids. The theoretical molecular mass of mature Mj-sty was 8.693 kDa with an isoelectric point (pI) of 4.79. A proline-rich N-terminal region and a C-terminal region contained 13 cysteine residues were identified. Genomic sequence analysis with respect to its cDNA showed that Mj-sty was organized into two exons interrupted by one intron. Tissue-specific expression revealed that Mj-sty was mainly transcribed in gills and hemocytes. Expression of Mj-sty in early developmental stages demonstrated that Mj-sty mRNA were present from fertilized eggs to post-larvae of 17 days (PL17), and the expression levels showed a significant variation in different developmental stages. After challenge of white spot syndrome virus (WSSV), the time-dependent expression pattern of Mj-sty in both gills and hepatopancrease showed down-regulation at the early hours of infection, subsequently up-regulation and down-regulation, and then up-regulation at the end hours to almost the half of the controls. The results indicate that Mj-sty is potentially involved in the ontogenesis and immune responses against WSSV.

Characterization of a novel piscidin-like antimicrobial peptide from Pseudosciaena crocea and its immune response to Cryptocaryon irritans.

Piscidins, important components of the innate (nonspecific) immunity system in fish, have potent, broad-spectrum antimicrobial and antiparasitic activities. In this study, we reported a novel antimicrobial cationic peptide from Pseudosciaena crocea. Although this peptide exhibited a genomic (3 exons and 2 introns) and propeptide (signal peptide, mature peptide and prodomain) organization, conserved signal peptide (22 amino acids) and consensus motif I-X5-H-X4-I-H identical to the reported fish piscidins, Pc-pis showed a relatively low overall conservation with other known piscidins, which was obviously embodied in the amino acid composition of the peptide. Pc-pis is strikingly rich in glycine residues (27.3%), which disrupted the amphipathic structure of the peptide. Relative quantitative real-time PCR revealed that Pc-pis is a typically gill-expressed peptide. The sequence analysis, structural features and tissue distribution suggested that Pc-pis was genetically related to the piscidins family and might be a novel piscidin-like antimicrobial peptide. Quantitative PCR analysis revealed that the expression of Pc-pis in the spleen, head-kidney, liver, intestine, skin and gill could be regulated during Cryptocaryon irritans infection and post C. irritans falling off, implicating a role for Pc-pis in immune defense against C. irritans and secondary bacterial infections. Synthetic Pc-pis exhibited broad-spectrum activity against bacteria, fungi and C. irritans in parasitic stages. These results provided the first evidence of piscidins antiparasitic activity against marine fish ectoparasites C. irritants trophonts and further indicated that Pc-pis might be an important component of the P. crocea innate immune system against C. irritans and secondary bacterial infections. Thus, these data provided new insights into P. crocea innate immunity against external protozoan parasite and microbial infections and facilitate the evaluation of Pc-pis as a therapeutic agent against pathogen invasion.