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Miiuy croaker, Miichthys miiuy is an ecologically important teleost species which is widely distributed in southeast coast of China. In this study, we present a chromosomal-scale genome assembly of the miiuy croaker which is an important Sciaenidae fish and economical species. We adopted Oxford Nanopore and Hi-C sequencing techniques to achieve an assembly with high accuracy and completeness. The investigation of genome characteristic and functional features may provide insights into the study of phylogenetic diversity of miiuy croaker. This study can also be beneficial to improve molecular assisted breeding techniques. Moreover, it can be a great resource to better conduct further study of other sciaenids.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) serves as a critical factor in the initiation of the NF-κB pathway and holds significance in modulating the inflammatory response. However, the regulatory mechanism of TRAF6 in teleost fish is poorly understood. We demonstrate Miichthys miiuy nucleoporin 43 (mm-nup43), a member of the nuclear pore complex (NPC), is a negative regulator of the NF-κB pathway and inhibits the expression of inflammatory cytokines. Mechanistically, mm-nup43 interacts with TRAF6 and inhibits it via the ubiquitin-proteasome pathway. Meanwhile, mm-nup43 decreased p65 phosphorylation, thereby negatively regulating NF-κB signaling. In summary, these results classify that mm-nup43 inhibits the TRAF6-mediated NF-κB pathway and prevents excessive inflammation.

The blackspotted croaker (Protonibea diacanthus) is an endangered coastal marine fish. It is also a valuable species that is cultured on the southeast coast of China. While some genetic studies have been conducted to protect this species, genomic resources are lacking. Here, we report a chromosome-scale assembly of P. diacanthus genome by high-depth genome sequencing, assembly, and annotation. The genome scale was 635.69 Mb with contig and scaffold N50 length of 3.33 Mb and 25.60 Mb, respectively. Hi-C scaffolding of the genome resulted in 24 chromosomes of 94.15% total genome. We predicted 23,971 protein-coding genes. In addition, we constructed a phylogenetic tree using 2755 single-copy gene families and identified 462 unique gene families in P. diacanthus genome compared to three other sciaenids. What's more, from the analysis of gene families, we found that several gene families related to innate immunity were significantly expanded in the blackspotted croaker genome compared to other teleost genomes. The high-quality genome can improve our understanding of the molecular mechanisms behind economically valuable traits and provide insights into characteristics of the immune system.

Mediator of IRF3 activation (MITA) is a significant signal adaptor in the retinoic acid-inducible gene-I like receptor (RLR) signaling pathway and plays an important role in the innate immune system. As a transcription factor, nuclear factor kappa B (NF-κB) can be available in many signaling pathways including the RLR signaling pathway and relative to biological processes like immune responses. In this study, it is determined that IRF4b and IRF8 can have a negative effect on NF-κB signaling pathway mediated by MITA in fish. Firstly, it is found that IRF4b and IRF8 have an inhibitory function on MITA-mediated NF-κB signaling pathway. It is interesting that IRF4b and IRF8 have similar functions to achieve precise downregulated and the degradation of MITA through the ubiquitin-proteasome pathway. IRF is taken as the core domain of IRF4b or IRF8 for the downregulation to MITA. This study provides data on MITA-mediated NF-κB signaling pathway in teleost fish and provides new insights into the regulatory mechanism in fish immune system.

Upon recognition of bacterial or viral components by Toll-like receptors (TLRs), cells could be activated to induce a series of reactions to produce inflammatory cytokines, type I interferon (IFN), and IFN stimulating genes (ISG). MicroRNAs (miRNAs) are an important regulatory molecules that are widely involved in the regulatory networks of mammalian inflammation and immune responses; however, in lower vertebrates, the regulatory network of miRNA-mediated immune responses is poorly understood. Here, we report two miRNAs form Miichthys miiuy, namely, miR-181b-2 and miR-21-1, that play a negative role in host antiviral and antibacterial immunity. We found that miR-181b-2 and miR-21-1 are abundantly expressed in gram-negative bacteria, as well as RNA rhabdovirus infection. Inducible miR-181b-2 and miR-21-1 suppress the production of inflammatory cytokines and type I IFN by targeting TRIF, thereby avoiding excessive inflammation. We further revealed that miR-181b-2 and miR-21-1 modulate antibacterial and antiviral immunity through the TRIF-mediated NF-κB and IRF3 signaling pathways. The overall results indicate that miR-181b-2 and miR-21-1 act as negative feedback regulators and participate in host antibacterial and antiviral immune responses; this finding could provide information for a deeper understanding of the resistance of lower vertebrates to the invasion of pathogens and to avoidance of excessive immunity.

Parupeneus chrysopleuron were under threat of climate change and unregulated fishing. For protecting the fish, complete mitochondrial genome (mitogenome) of P. chrysopleuron was firstly determined. The mitogenome is 17,397 nucleotides, including 38 genes. The gene arrangement of the P. chrysopleuron mitogenome is different from most of bony fishes. Investigating the mitochondrial gene arrangement pattern found that the tRNAIle and tRNAGln, followed by tRNAMe. Observing four notable non-coding regions, a non-coding region of 814 bp was identified as control region based on its location and central conserved sequence blocks (CSB-1 and CSB-2) were recognized.

Pathogen infection can cause the production of inflammatory cytokines, which are key mediators that cause the host’s innate immune response. Therefore, proper regulation of immune genes associated with inflammation is essential for immune response. Among them, microRNAs (miRNAs) as gene regulator have been widely reported to be involved in the innate immune response of mammals. However, the regulatory network in which miRNAs are involved in the development of inflammation is largely unknown in lower vertebrates. Here, we identified two miRNAs from miiuy croaker (Miichthys miiuy), miR-210 and miR-3570, which play a negative regulatory role in host antibacterial immunity. We found that the expressions of miR-210 and miR-3570 were significantly upregulated under the stimulation of Gram-negative bacterium vibrio harveyi and LPS (lipopolysaccharide). Induced miR-210 and miR-3570 inhibit inflammatory cytokine production by targeting RIPK2, thereby avoiding excessive inflammation. In particular, we found that miR-210 and miR-3570 negatively regulate antimicrobial immunity by regulating the RIPK2-mediated NF-κB signaling pathway. The collective results indicated that both miRNAs are used as negative feedback regulators to regulate RIPK2-mediated NF-κB signaling pathway and thus play a regulatory role in bacteria-induced inflammatory response.

The innate immune organs and cells detect the invasion of pathogenic microorganisms, which trigger the innate immune response. A proper immune response can protect the organisms from pathogen invasion. However, excessive immunity can destroy immune homeostasis, leading to uncontrolled inflammation or pathogen transmission. Evidence shows that the miRNA-mediated immune regulatory network in mammals has had a significant impact, but the antibacterial and antiviral responses involved in miRNAs need to be further studied in lower vertebrates. Here, we report that miR-2187 as a negative regulator playing a critical role in the antiviral and antibacterial response of miiuy croaker. We find that pathogens such as Vibrio anguillarum and Siniperca chuatsi rhabdovirus (SCRV) can up-regulate the expression of miR-2187. Elevated miR-2187 is capable of reducing the production of inflammatory factors and antiviral genes by targeting TRAF6, thereby avoiding excessive inflammatory response. Furthermore, we proved that miR-2187 modulates innate immunity through TRAF6-mediated NF-κB and IRF3 signaling pathways. The above results indicate that miR-2187 acts as an immune inhibitor involved in host antibacterial and antiviral responses, thus enriching the immune regulatory network of the interaction between host and pathogen in lower vertebrates.

Long noncoding RNAs (lncRNAs) function as micro regulators to impact gene expression after multiple pathogen infections, which have been largely studied in the last few years. Although lncRNA studies on lower vertebrates have received less attention than those on mammals, current studies suggest that lncRNA plays an important role in the immune response of fish to pathogen infections. Here, we studied the effect of MIR122HG as the host gene of miR-122 and indirectly negatively regulate MAVS-mediated antiviral immune responses in miiuy croaker (Miichthysmiiuy). We found that poly(I:C) significantly increases the host MIR122HG expression. The increased MIR122HG expression inhibited the production of the antiviral immune-related genes IFN-1, ISG15 and Viperin upon SCRV treatment. In addition, MIR122HG can act as a pivotally negative regulator involved in the MAVS-mediated NF-κB and IRF3 signaling pathways, which can effectively avoid an excessive immune response. Additionally, we found that MIR122HG can promote the replication of SCRV. Our study provides evidence about the involvement of lncRNAs in the antiviral immune response of fish and broadens the understanding of the function of lncRNAs as a precursor miRNA in teleost fish.

Uranoscopus tosae is a marine fish which some wild populations are in danger, and need urgent conservation. In this study, the complete mitochondrial genome of the U. tosae which is 17,286 base pairs in length, and contained 13 protein-coding genes and 2 ribosomal RNA genes, 22 tRNA genes and 2 main non-coding regions was firstly determined. Most of the genes of U. tosae were encoded on the H-strand, while the ND6 and eight tRNA (Gln, Ala, Asn, Cys, Tyr, Ser (UCN), Glu and Pro) genes were encoded on the L-strand. In addition, the complete mitochondrial genome of U. tosae was used to construct phylogenetic tree, which shows that U. tosae clustered with the species of Howellidae. The mitochondrial genome of U. tosae provides important data for studying on population genetic diversity and genetic conservation.

The Semicossyphus reticulatus is a marine fish species belongs to the family Labridae. To understand the phylogenetic relationship of S. reticulatus in teleost, we firstly determined the complete mitochondrial genome of S. reticulatus. The entire mitochondrial genome (mtDNA) of S. reticulatus is 16610 bp in length, including 13 protein-coding genes and 2 ribosomal RNA genes, 22 tRNAs and 2 main non-coding regions. The gene arrangement, base composition, and tRNA structures of the mtDNA of S. reticulatus is consistent with those of other teleost. The S. reticulatus mtDNA was used to construct phylogenetic tree, which shows that S. reticulatus is clustered with the fishes of the order Perciformes. We expect that the availability of mtDNA of S. reticulatus will facilitate the further investigations of the taxonomic resolution, biogeography and molecular systematic.

Caranx equula is an important marine fish which due to its commercial values. However, some wild populations of C. equula are in danger because of the overfishing and environmental pollution. In this study, the complete mitochondrial genome of C. equula was firstly determined. The complete mitochondrial genome is 16,607 base pairs in length, and contained 13 protein-coding genes and 2 ribosomal RNA genes, 22 tRNA genes and 2 main non-coding regions. Overall A + T content was 52.9%. In addition, a phylogenetic tree was constructed using the complete mitochondrial genome and showed that C. equula clustered in a clade and formed a sister relationship with Caranx ignobilis belonged to the family of Carangidae.

Red bigeye, Priacanthus macracanthus, a species in the family Priacanthidae was added to the seafood red list by Greenpeace International due to the decline in its abundance and overfishing. To understand the phylogenetic relationship of Red bigeye in teleost, we determined the complete mitochondrial genome of Red bigeye. The complete mitochondrial genome is 17,003bp in length, including 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 2 main non-coding regions. Phylogenetic tree shows that Red bigeye is clustered with the fishes of the order Perciformes.

To understand the phylogenetic position of Larimichthys polyactis within the family Sciaenidae and the phylogeny of this family, the organization of the mitochondrial genome of small yellow croaker was determined herein. The complete, 16,470 bp long, mitochondrial genome contains 37 mitochondrial genes (13 protein-coding, 2 ribosomal RNA and 22 transfer RNA genes), as well as a control region (CR), as in other bony fishes. Comparative analysis of initiation/termination codon usage in mitochondrial protein-coding genes of Percoidei species, indicated that COI in Sciaenidae entails an ATG/AGA codon usage different from other Percoidei fishes, where absence of a typical conserved domain or motif in the control regions is common. Partitioned Bayesian analysis of 618 bp of COI sequences data were used to infer the phylogenetic relationships within the family Sciaenidae. An improvement in harmonic mean -lnL was observed when specific models and parameter estimates were assumed for partitions of the total data. The phylogenetic analyses did not support the monophyly of Otolithes, Argyrosomus, and Argyrosominae. L. polyactis was found to be most closely related to Collichthys niveatus, whereby, according to molecular systematics studies, the relationships within the subfamily Pseudosciaenidae should be reconsidered.

The Chinese salamander (Hynobius chinensis), an endangered amphibian species of salamander endemic to China, has attracted much attention because of its value of studying paleontology evolutionary history and decreasing population size. Despite increasing interest in the Hynobius chinensis genome, genomic resources for the species are still very limited. A comprehensive transcriptome of Hynobius chinensis, which will provide a resource for genome annotation, candidate genes identification and molecular marker development should be generated to supplement it.We performed a de novo assembly of Hynobius chinensis transcriptome by Illumina sequencing. A total of 148,510 nonredundant unigenes with an average length of approximately 580 bp were obtained. In all, 60,388 (40.66%) unigenes showed homologous matches in at least one database and 33,537 (22.58%) unigenes were annotated by all four databases. In total, 41,553 unigenes were categorized into 62 sub-categories by BLAST2GO search, and 19,468 transcripts were assigned to 140 KEGG pathways. A large number of unigenes involved in immune system, local adaptation, reproduction and sex determination were identified, as well as 31,982 simple sequence repeats (SSRs) and 460,923 putative single nucleotide polymorphisms (SNPs).This dataset represents the first transcriptome analysis of the Chinese salamander (Hynobius chinensis), an endangered species, to be also the first time of hynobiidae. The transcriptome will provide valuable resource for further research in discovery of new genes, protection of population, adaptive evolution and survey of various pathways, as well as development of molecule markers in Chinese salamander; and reference information for closely related species.

The Hapalogenys genus was the most controversial and problematic in phylogenetic position of Percoidei. In this study, we rechecked the taxonomic status of Hapalogenys in Percoidei using complete mitochondrial genome data. We purposefully added a new complete mitochondrial sequence from chosen species of Hapalogenys and conducted phylogenetic analyses using a large complete mitochondrial data set. The resultant tree topologies were congruent from partitioned Bayesian and Maximum-likelihood methods. The results indicated that Hapalogenys was distantly related to Haemulidae and could be removed from Haemulidae. The results supported the Hapalogeny was upgraded to a family rank titled Hapalogenyidae, and it should be recognized in a separate family of Hapalogenyidae. A relaxed molecular-clock Bayesian analysis indicated that the divergence times of Perciformes groups had a much older than the available old fossil records. The origin of the common ancestral lineage of Perciformes fish was estimated in the late Jurassic about 149 Myr ago.

Transferrin (TF) is a protein that plays a central role in iron metabolism. This protein is associated with the innate immune system, which is responsible for disease defense responses after bacterial infection. The clear link between TF and the immune defense mechanism has led researchers to consider TF as a candidate gene for disease resistance. In this study, the Miichthys miiuy (miiuy croaker) TF gene (MIMI-TF) was cloned and characterized. The gene structure consisted of a coding region of 2070 nucleotides divided into 17 exons, as well as a non-coding region that included 16 introns and spans 6757 nucleotides. The deduced MIMI-TF protein consisted of 689 amino acids that comprised a signal peptide and two lobes (N- and C-lobes). MIMI-TF expression was significantly up-regulated after infection with Vibrio anguillarum. A series of model tests implemented in the CODEML program showed that TF underwent a complex evolutionary process. Branch-site models revealed that vertebrate TF was vastly different from that of invertebrates, and that the TF of the ancestors of aquatic and terrestrial organisms underwent different selection pressures. The site models detected 10 positively selected sites in extant TF genes. One site was located in the cleft between the N1 and N2 domains and was expected to affect the capability of TF to bind to or release iron indirectly. In addition, eight sites were found near the TF exterior. Two of these sites, which could have evolved from the competition for iron between pathogenic bacteria and TF, were located in potential pathogen-binding domains. Our results could be used to further investigate the function of TF and the selective mechanisms involved.

Hepcidin antimicrobial peptide (HAMP) is a small cysteine-rich peptide and a key molecule of the innate immune system against bacterial infections. Molecular cloning and genomic characterization of HAMP gene in the miiuy croaker (Miichthys miiuy) were reported in this study. The miiuy croaker HAMP was predicted to encode a prepropeptide of 99 amino acids, a tentative RX(K/R)R cleavage motif and eight characteristic cysteine residues were also identified. The gene organization is also similar to corresponding genes in mammals and fish consisting of three exons and two introns. Sequence polymorphism analysis showed that only two different sequences were identified and encoded two proteins in six individuals. As reported for most other species, the expression level was highest in liver and an up-regulation of transcription was seen in spleen, intestine and kidney examined at 24 h after injection of pathogenic bacteria, Vibrio anguillarum, the expression pattern implied that miiuy croaker HAMP is an important component of the first line defense against invading pathogens. In addition, we report on the underlying mechanism that maintains sequences diversity among fish and mammalian species, respectively. A series of site-model tests implemented in the CODEML program revealed that moderate positive Darwinian selection is likely to cause the molecular evolution in the fish HAMP2 genes and it also showed that the fish HAMP1 genes and HAMP2 genes under different selection pressures.

BACKGROUND: The third complement component (C3) is a central protein of the complement system conserved from fish to mammals. It also showed distinct characteristics in different animal groups. Striking features of the fish complement system were unveiled, including prominent levels of extrahepatic expression and isotypic diversity of the complement components. The evidences of the involvement of complement system in the enhancement of B and T cell responses found in mammals indicated that the complement system also serves as a bridge between the innate and adaptive responses. For the reasons mentioned above, it is interesting to explore the evolutionary process of C3 genes and to investigate whether the huge differences between aquatic and terrestrial environments affected the C3 evolution between fish and mammals. METHODOLOGY/PRINCIPAL FINDINGS: Analysis revealed that these two groups of animals had experienced different evolution patterns. The mammalian C3 genes were under purifying selection pressure while the positive selection pressure was detected in fish C3 genes. Three periods of positive selection events of C3 genes were also detected. Two happened on the ancestral lineages to all vertebrates and mammals, respectively, one happened on early period of fish evolutionary history. CONCLUSIONS/SIGNIFICANCE: Three periods of positive selection events had happened on C3 genes during history and the fish and mammals C3 genes experience different evolutionary patterns for their distinct living environments.

Major histocompatibility complex (MHC) has a central role in the adaptive immune system by presenting foreign peptide to the T-cell receptor. In order to study the molecular function and genomic characteristic of class II genes in teleost, the full lengths of MHC class IIA and IIB cDNA and genomic sequence were cloned from miiuy croaker (Miichthys miiuy). As in other teleost, four exons and three introns were identified in miiuy croaker class IIA gene; but the difference is that six exons and five introns were identified in the miiuy croaker class IIB gene. The deduced amino acid sequence of class IIA and class IIB had 26.3-85.7% and 11.0-88.8% identity with those of mammal and teleost, respectively. Real-time quantitative RT-PCR demonstrated that the MHC class IIA and IIB were ubiquitously expressed in ten normal tissues; expression levels of MHC genes were found first upregulated and then downregulated, and finally by a recovery to normal level throughout the pathogenic bacteria infection process. In addition, we report on the underlying mechanism that maintains sequences diversity among many fish species. A series of site-model tests implemented in the CODEML program revealed that positive Darwinian selection is likely the cause of the molecular evolution in the fish MHC class II genes.