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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.

Sciaenops ocellatus, also known as red drum, is a valuable commercial fish that is widely cultured in China. In recent years, while the genetic research and molecular breeding on S. ocellatus has been widely performed, whole-genome resources are lacking. In our study, we assembled the S. ocellatus genome by high-quality genome sequencing, followed by complete annotation. The size of final genome was 686.62 Mb and N50 length of contig and scaffold is 99.71 Kb and 25.62 Mb, respectively. Using the Hi-C technology, we obtained 24 pseudochromosomes containing 87.82% of the total scaffolds. We annotated 20,053 protein-coding genes in S. ocellatus genome, of which 99.93% were homologous to proteins in different database. Totally, we detected 133.54 Mb repeat sequences, which accounts for 19.45% of the assembly. According to the phylogenetic analysis, S. ocellatus is most closed to L. croea with a divergence time about 30 million years ago. We assembled a high-quality genome that will be a valuable resource to further study the biology of S. ocellatus and will inform how to breed the red drum to enhance its economic traits.

Viral infection induces the initiation of antiviral effectors and cytokines which are critical mediators of innate antiviral responses. The critical molecular determinants are responsible for triggering an appropriate immune response. Long noncoding RNAs (lncRNAs) have emerged as new gene modulators involved in various biological processes, while how lncRNAs operate in lower vertebrates are still unknown. Here, we discover a long noncoding RNA, termed antiviral-associated long noncoding RNA (AANCR), as a novel regulator for innate antiviral responses in teleost fish. The results indicate that fish MITA plays an essential role in host antiviral responses and inhibition of Siniperca chuatsi rhabdovirus (SCRV) production. miR-210 reduces MITA expression and suppress MITA-mediated antiviral responses, which may help viruses evade host antiviral responses. Further, AANCR functions as a competing endogenous RNA (ceRNA) for miR-210 to control protein abundance of MITA, thereby inhibiting SCRV replication and promoting antiviral responses. Our data not only shed new light on understanding the function role of lncRNA in biological processes in teleost fish, but confirmed the hypothesis that ceRNA networks exist widely in vertebrates.

The suppressor of cytokine signaling (SOCS) was first described as inhibitors of cytokine signaling. The SOCS1, as a number of SOCS family, is an important negative regulator in the IFN signaling pathways in mammals. While data on functional characterization of SOCS1 in lower vertebrates are limited. In this study, we identified and characterized the full length SOCS1b gene of miiuy croaker (Miichthys miiuy). The sequence alignment analysis results showed that miiuy croaker SOCS1b (mmSOCS1b) have only a conserved SH2 domain that is similar to other vertebrates. To further study the functions of mmSOCS1b, we identified and determined its potential ability to perceive poly (I:C) stimulation. Stimulation experiments with poly (I:C) showed the significantly upregulated expression of mmSOCS1b in crucial immune-related tissues of spleen and kidney, indicating that mmSOCS1b might participate in the immune responses. Furthermore, the immunofluorescence assay indicated that mmSOCS1b present in the cytoplasmic of HeLa cells. In addition, mmSOCS1b could inhibit IFNα or IFNγ-induced ISRE reporter gene. In a word, we systematically and comprehensively analyzed the characterizations and functions of mmSOCS1b, which not only enriches the current knowledge of SOCS in IFN signaling regulation but also offer the basis for future research of fish SOCS family.

MDA5 is a member of retinoic acid-inducible gene I (RIG-I)-like receptors (RLR receptors), which may play a crucial role in the immune regulation process. Recently, microRNAs (miRNAs) have been shown to act as an important regulator in the RLRs signaling pathway. Additionally, the MDA5 gene, as a significant cytosolic pathogen recognition receptor (PRR), its characteristics and functions have been extensively investigated, while less research has been done on the mechanisms of MDA5-miRNA mediated gene regulation. In this study, the evolution and functional characterization of MDA5 from miiuy croaker (mmiMDA5) were characterized. Comparative genomic analysis demonstrated that the ascidiacea and superclass do not have the MDA5 gene in the process of evolution. MDA5 contains four structural domains: CARD, ResIII, Helicase C, and RIG-I C-RD. The MDA5 was ubiquitously expressed in all tested miiuy croaker tissues. Moreover, the expressions were significantly up-regulated after stimulation with poly (I: C), which indicated that MDA5 might be involved in the antiviral immune response. The bioinformatics predicted programs have indicated that miR-203 has a direct negative regulatory effect on MDA5 in miiuy croaker. Furthermore, the dual-luciferase reporter assay have showed that miR-203 was the direct negative regulator of MDA5 in miiuy croaker. This study is the first to demonstrate that miRNA can suppress cytokines by regulating the RLR signaling pathway in teleost fish, providing some new ideas for studying miRNA-mediated regulation of immune responses in mammals.

Parupeneus chrysopleuron were under threat of climate change and unregulated fishing. For protecting the fish, complete mitochondrial genome (mitogenome) of P. chrysopleuron was firstly det...

Innate immune response is an important response mechanism for the host to achieve self-protection, and it plays an important role in identifying pathogens and resisting pathogen invasion. Growing evidences have shown that microRNA functions as a crucial regulator involved in the host innate immune response. In this study, the regulations of miR-1388-5p to regulate NF-κB signaling pathways via targeting the IRAK1 gene was studied in miiuy croaker. First, through bioinformatics software prediction, we found that IRAK1 is the direct target of miR-1388-5p, and then the prediction results were verified by using dual-luciferase assays. Next, we found that both miR-1388-5p mimics and pre-miR-1388 plasmids inhibit IRAK1 expression by complementing the seed sequence in the 3'-untranslated region (3'-UTR) of IRAK1. Finally, we observed that miR-1388-5p could negatively regulate NF-κB pathways through targeting IRAK1. These results provide new insights into the function of miR-1388-5p in fish innate immunity, meanwhile enriching miRNA-mediated regulatory networks.

Interferon regulatory factors (IRFs) are transcription factors which play important roles in regulating the expression of type I interferons (IFNs) and IFN-stimulated genes. IRF9 is one of the IRF family gene members which belongs to the IRF4 subfamily. Mammalian IRF9 has been known to be involved in antiviral responses as the DNA sequence recognition subunit of IFN-stimulated gene factor 3 (ISGF3) complex. In fish, only a few studies investigated the characteristics of IRF9 and the role in IFN signaling. In this study, we identified the IRF9 gene from miiuy croaker (mmiIRF9) and studied its feature and function. Sequence analysis showed the similarity of mmiIRF9 and other fish IRF9 genes. Structural and syntenic analysis showed the conservatism in fish IRF9 genes. The result of expression analysis in normal tissues and infected tissues and macrophages showed that mmiIRF9 expressed in all tested normal tissues and up-regulated expression in liver, kidney and macrophages after stimulated with poly(I:C). Luciferase reporter assays demonstrated the mmiIRF9 can induced IFNα and IFNβ luciferase reporters and the cellular localization of mmiIRF9 was mainly distributed in the cytoplasm in Hela cells. Furthermore, the evolutionary analysis of IRF4 subfamily showed the IRF4 and IRF8 may be the most ancient and conservative genes in the evolution of this subfamily.

Interleukin (IL)-1β is a prototypical proinflammatory cytokine that belongs to the IL-1 family. This cytokine possesses two receptor types, namely, IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII). IL-1RI, is an IL-1 receptor that plays a crucial role in immune responses and IL-1RII is a decoy receptor for IL-1β signaling inhibitors in mammals. IL-1β, together with its two types of receptors, has been characterized in mammals and implicated in immunity. However, IL-1β and IL-1 receptors in teleost species have been rarely investigated. In this study three genes, namely, IL-1β, IL-1RI, and IL-1RII, were identified and characterized from miiuy croaker. Structural and comparative analysis revealed that miiuy croaker IL-1β, IL-1RI and IL-1RII, particularly their functional domains, were conservative in most of the species. Simultaneously, synteny phylogenetic analysis indicated that IL-1β and IL-18 were widely distributed in vertebrates and hence might be the ancestors of the IL-1 family. Challenge experiment demonstrated that IL-1β, IL-1RI and IL-1RII expression in miiuy croaker was induced by LPS and poly (I:C). IL-1RI expression was also induced by the overexpressed miiuy croaker IL-1β protein which in cell supernatant, whereas IL-1RII was not induced.

The double-Ig-IL-1R related molecule (DIGIRR) is a member of the TIR (Toll -Interleukin-1 receptor) superfamily and plays an important role in the immune system, it is also as a negative regulator of the IL-1 signaling pathway. In this study, we identified and characterized the miiuy croaker DIGIRR (mmi-DIGIRR) gene. The results of gene structure analysis indicated that there were differences between the mmi-DIGIRR and mammalian SIGIRR, which there were two immunoglobulin (Ig) domains contained in extracellular region of mmi-DIGIRR. Sequence alignment analysis showed that fish DIGIRR shared some conserved sequences with other vertebrates and the evolution was relatively conservative. In order to further validate the function of mmi-DIGIRR and its expression levels in various tissues of fish, qRT-PCR has been conducted. The results showed DIGIRR has significant expression levels in liver, skin and muscle while expression levels in heart are low. The LPS-induced NF-κB activation was inhibited by overexpression of DIGIRR significantly. In conclusion, the evolution and function of mmi-DIGIRR were comprehensively analyzed in this study, which would provide a theoretical basis for the future research of fish DIGIRR.

Plant endophytes are microbes that colonize plant internal tissues and are ubiquitously associated with plants. In this study, seven endophytic bacterial strains, 665L2, 725L2, 725R2, 92R2, 728R3, 728R4 and 2416T3, were isolated from seeds of five healthy maize varieties (Zea mays L.) and all identified as Bacillus velezensis by polyphasic taxonomy based on 16S rRNA and gyrA gene phylogenetic analysis. In addition, according to the genotyping results from random amplified polymorphic DNA (RAPD), 665L2, 725L2, 725R2 and 92R2 belonged to the same strain, while 728R3 and 2416T3 belonged to another strain. Pathogenic fungal strains 4, 5 and 6 were isolated from three diseased maize varieties (Zea mays L.), and they were identified as Talaromyces funiculosus, Penicillium oxalicum and Fusarium verticillioides, respectively, by polyphasic taxonomy based on morphological identification, ITS rDNA and bio-control gene phylogenetic analyses. Seven endophytic bacterial Bacillus velezensis strains had favourable antagonistic activity, and antagonistic testing was carried out against the three pathogenic strains, Talaromyces funiculosus 4, Penicillium oxalicum 5 and Fusarium verticillioides 6. Biological control lipopeptide antibiotic genes (bioA, bmyB, ituC, fenD, srfAA, srfAB, yngG and yndJ) were amplified using specific primers, and they were found in the seven endophytic bacterial Bacillus velezensis strains. This study provides a scientific basis for future research on the use of resistant endophytic bacterial resources to enhance crop production.

NOD-like receptors (NLRs) are essential intracellular pattern-recognition receptors that respond to pathogens and regulate innate immunity. NLRs include three distinct subfamilies: NLR-A, NLR-B and NLR-C, thereinto, NLR-C as a large subfamily is unique to bony fish and little research about it has been done. In the current study, we identified the members of NLR-B and NLR-C subfamilies containing 2 and 48 genes respectively in miiuy croaker. Compared with other teleosts except for zebrafish, NLR-C subfamily genes occurred expansion in miiuy croaker. The gene expansions of NLR-C subfamily may illustrate adaptive genome evolution in response to specific aquatic environments. Structural analysis showed that the N-terminus of NLR-C subfamily receptors has different characteristics of the domains including RING domain, FISNA domain or PYRIN domain. Interestingly, the C-terminus of 18 NLR-C subfamily members contains an extra B30.2 domain (named NLR-B30.2 genes) which plays an important role in antiviral immune recognition. Simultaneously, molecular evolutionary analysis indicated that the positively sites in miiuy croaker are mainly located in NACHT domain which was the vital region for signal transduction in immune response. Significantly, pathogens challenge in spleen and macrophages demonstrated that NLR-B30.2 genes exhibited more sensitive response to virus than bacteria, suggesting these genes play enhanced roles in innate antiviral immunity, which may represent a new family used for antiviral infection.

Increasing evidence suggests important roles for long noncoding RNAs (lncRNAs) as new gene modulators involved in various biological processes. However, the function roles of lncRNAs in lower vertebrates are still unknown. Here, we firstly identify a lncRNA, named MAVS antiviral-related lncRNA (MARL), as a key regulator for antiviral immunity in teleost fish. The results indicate that fish MAVS play essential roles in host antiviral responses and inhibition of Siniperca chuatsi rhabdovirus (SCRV) replication. miR-122 reduces MAVS expression and suppress MAVS-mediated antiviral responses, which may help viruses evade host antiviral responses. Further, MARL functions as a competing endogenous RNA (ceRNA) for miR-122 to control protein abundance of MAVS, thereby inhibiting SCRV replication and promoting antiviral responses. Our data not only shed new light on understanding the function role of lncRNA in biological processes in lower vertebrates, but confirmed the hypothesis that ceRNA regulatory networks exist widely in vertebrates., Author summary Increasing evidence indicates that lncRNAs participate in the regulation of various biological processes, especially innate and adaptive immunity. However, the relationship between lncRNAs and host antiviral responses remains largely unknown, particularly in lower vertebrates. Our results provided the first direct evidence that a lncRNA, termed MAVS antiviral-related lncRNA (MARL), acts as a key regulator for antiviral immunity in lower vertebrates. lncRNAs have been identified to function as competing endogenous RNAs (ceRNAs) and cross-talk with mRNAs by competing shared for miRNAs. Such ceRNAs regulate the distribution of miRNA molecules on their targets and thereby apply an additional level of post-transcriptional regulation. In our study, MARL functions as a ceRNA for miR-122 to control protein abundance of fish MAVS, thereby inhibiting virus replication and promoting antiviral responses. This is the first study to demonstrate ceRNA regulatory networks existing in lower vertebrates, which can provide new insights into understanding the effects of lncRNAs on host-virus interactions.

Ariosoma meeki (A. meeki) is a demersal and carnivorous fish species belongs to the family Congridae. Some wild populations of A. meeki are in danger because of the overfishing and environmental pollution. In order to better understand the germplasm resources, the complete mitochondrial genome of A. meeki was firstly determined in this study. The complete mitochondrial genome is 16,404 nucleotides, comprising 12 protein-coding genes, 2 ribosomal RNA genes, 20 tRNA genes, and 2 main non-coding regions, but ND6 and two tRNA genes were not found in the A. meeki mitogenome. The A. meeki mitogenome was currently the first member of Ariosoma genus with gene and tRNA deletion. In addition, phylogenetic analysis result demonstrated that A. meeki and A. shiroanago were clustered in a clade and formed a sister relationship.

Four Gram-stain positive bacterial strains, designated as 4R1(T), 4R9, 4L13 and 4L18, isolated from seeds of hybrid maize (Zea mays L., Jingke 968), were investigated using a polyphasic taxonomic approach. The cells were found to be facultatively aerobic, motile, spore-forming and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates should be recognised as a species of the genus Paenibacillus, with two close neighbours being Paenibacillus nicotianae YIM h-19(T) (98.41 % similarity) and Paenibacillus hordei RH-N24(T) (98.37 %). The DNA G+C content of strain 4R1(T) was determined to be 51.6 mol %. Its polar lipid profile was found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified lipid. The predominant respiratory quinone was identified as MK-7 and the major fatty acids were found to be anteiso-C15:0, anteiso-C12:0, anteiso-C13:0 and anteiso-C11:0. Strains 4R1(T), 4R9, 4L13 and 4L18 were clearly distinguished from the reference type strains using phylogenetic analysis, DNA-DNA hybridization and a range of physiological and biochemical characteristics. It is evident from the genotypic and phenotypic data that strains 4R1(T), 4R9, 4L13 and 4L18 represent a novel species of the genus Paenibacillus, for which the name Paenibacillus chinensis sp. nov. is proposed. The type strain is 4R1(T) (=KCTC 33672(T) = CICC 23864(T)).

Akirins, which are highly conserved nuclear proteins, are present throughout the metazoan and regulate innate immunity, embryogenesis, myogenesis, and carcinogenesis. This study reports all akirin genes from miiuy croaker and analyzes comprehensively the akirin gene family combined with akirin genes from other species. A second nuclear localization signal (NLS) is observed in akirin2 homologues, which is not in akirin1 homologues in all teleosts and most other vertebrates. Thus, we deduced that the loss of second NLS in akirin1 homologues in teleosts likely occurred in an ancestor to all Osteichthyes after splitting with cartilaginous fish. Significantly, the akirin2(2) gene included six exons interrupted by five introns in the miiuy croaker, which may be caused by the intron insertion event as a novel evidence for the variation of akirin gene structure in some species. In addition, comparison of the genomic neighborhood genes of akirin1, akirin2(1), and akirin2(2) demonstrates a strong level of conserved synteny across the teleost classes, which further proved the deduction of Macqueen and Johnston 2009 that the produce of akirin paralogues can be attributed to whole-genome duplications and the loss of some akirin paralogues after genome duplications. Furthermore, akirin gene family members and relish gene are ubiquitously expressed across all tissues, and their expression levels are increased in three immune tissues after infection with Vibrio anguillarum. Combined with the expression patterns of LEAP-1 and LEAP-2 from miiuy croaker, an intricate network of co-regulation among family members is established. Thus, it is further proved that akirins acted in concert with the relish protein to induce the expression of a subset of downstream pathway elements in the NF-kB dependent signaling pathway.

The complement system, as one of the most sophisticated innate immune system, plays an important role in defense against invading microorganisms. The complement component C7 participates in the cytolytic phase of complement activation through a series of polymerization reactions with other terminal complement components. In this study, we derived two C7 genes from the whole genome of miiuy croaker which were the consequence of the fish-specific genome duplication. Our data showed that miiuy croaker C7-1 and C7-2 genes shared same structure domains. The analysis of gene synteny showed that high degree conserved of synteny was retained between miiuy croaker and other teleosts, and miiuy croaker had a relatively closer relationship with fugu. The expression of C7-1 and C7-2 in miiuy croaker healthy tissues revealed that they were ubiquitously expressed in all ten tested tissues. Besides, the immune response of C7-1 and C7-2 were different in spleen with Vibrio anguillarum, Staphylococcus aureus, poly I:C and LPS at 24 h post-injection, respectively. Furthermore, the expression patterns of C7-1 and C7-2 were different in liver, spleen and kidney after infected with V. anguillarum at different time-point. Evolutionary analysis showed that all the ancestral lineages underwent positive selection except for the ancestral lineages of fish C7-2, indicated that the ancestral lineages of fish C7-1 genes undertook more pressures than C7-2 in defense against the invading microorganisms. Meanwhile, a series of maximum likelihood methods were used to explore the evolutionary patterns on extant vertebrates' C7 genes. Three and one positive selection sites were found in extant mammalian C7 genes and fish C7-2 genes, but no positive selection site was found in extant fishes C7-1 genes. The result showed that extant fish C7-2 genes undertook more pressures compared with C7-1. In conclusion, fish C7-1 and C7-2 gene underwent different evolutionary patterns.

β2-Microglobulin (β2m) is related to major histocompatibility complex class I alpha chains, and forms cell-surface glycoproteins that mediate a variety of functions in immune defense. In general, β2m has no isoforms and is not polymorphic in higher vertebrates, but polymorphisms between different alleles have been found in some fish species. In this study, full-length β2m cDNA and genomic sequences were cloned from the miiuy croaker (Miichthys miiuy). The miiuy croaker β2m gene shares many of the same characteristics as other fish species. Three exons and two introns were identified in the miiuy croaker β2m gene; these genomic structural features are similar to those present in other fish. The deduced β2m amino acid sequence exhibited 34.7-90.1% identity with mammal and teleost β2m amino acid sequences. Sequence polymorphism analysis in six individuals identified three alleles that encoded two proteins, confirming that β2m polymorphisms exist in this species. Phylogenetic analysis elucidated the evolutionary history of the β2m protein among warm-blooded vertebrates and bony fish.

The suppressor of cytokine signaling 1 (SOCS1) is a crucial regulator in the immune systems of mammals, which functions classically as a negatively regulator in the IFN signaling pathways. However, data on functional characterization of SOCS1 in lower vertebrates is limited. In this study, we identified and characterized the full-length SOCS1a gene of miiuy croaker (Miichthys miiuy). The sequence analysis results showed that miiuy croaker SOCS1a (mmSOCS1a) shared some conserved motifs with other vertebrates. To further study the function of fish SOCS1, we identified mmSOCS1a and determined its potential ability to perceive poly(I:C) stimulation. Induction experiments with poly(I:C) indicated the significant expression levels of mmSOCS1a in liver and kidney. In addition, mmSOCS1a could inhibit poly(I:C)-induced or IFNs-induced ISRE reporter gene activation. In a word, we systematically and comprehensively analyzed evolution and function of mmSOCS1a, which will provide the basis for future research on fish SOCS family.

Interferon regulatory factor (IRF) family is a transcription factor family which plays an important role in the regulation of natural immunity and immune cell differentiation. IRF7 is important to regulate the response of type I interferon (IFN) to viral infection. Thus, more researches of the characteristic and functions of IRF7 should be done to get better understanding of the mechanisms underlying immune reactions. Here, the characterization of full-length cDNA of IRF7 was reported from miiuy croaker. Gene characterization analysis of mmiIRF7 showed conservative with other fish and inferred that the difference of tryptophan residues in IRF7 may occurred in the period of fish-specific genome duplication (FSGD) or earlier. Syntenic analysis of IRF7 showed that fish IRF7 had more highly conserved synteny than the higher vertebrates IRF7. Luciferase reporter assays result showed the ability of mmiIRF7 for activation of IFNα, IFNβ, IFNγ and ISRE luciferase reporter. In this study, we systematically and comprehensively analyzed evolution and function of mmiIRF7, which will provide the basis for future research on fish IRF family.

Leptin is a key harmone in regulating food intake, immune function and energy metabolism in vertebrates. Since it was first cloned in mouse (Zhang et al. 1994) , the leptin gene has received much attention and people started to research its function in energy homoeostasis and reproductive regulation (Campfiled et al. 1995). A large number of researchers have discovered that leptin is present in several fishes and it plays a similar role in regulation of food intake and energy metabolism in fishes as in mammals (Copeland et al. 2011). Although evidence of positive selection on the leptin gene has been documented in primates, seals and chiropterans (Yu et al. 2011), studies on evolution of leptin gene in fishes are fewer. Leptin, the product of the ob gene, was discovered in 1950 (Coleman 2010). The discovery was followed by intense research into the properties and physiological action of leptin. In initial studies, people considered that the primary functions of leptin were its influence on metabolic rate and mobilization of fat stores (Minokoshi and Kahn 2003). With further research, we now know that leptin is pleiotropic, exerting effects on reproduction, immune function, capillary growth and bone remodelling (Friedman 2009). In mammals, leptin acts on the hypothalamus which sends further signals to the brain, leading the brain to regulate food intake and metabolism (Pelleymounter et al. 1995). To our knowledge, mutations in the leptin genes were associated with a large number of hormonal and metabolic alterations (Kennedy 1953). Deficiency of leptin can cause obesity and diabetes in humans and mice (Strobel et al. 1998). Leptin genes of some fishes have been cloned and people have started to study their structure and biological functions of the protein (Murashita et al. 2008; Aguilar et al. 2010). Although leptin genes of several several species of teleosts have been cloned (Kurokawa et al. 2005; Huising et al. 2006; Ronnestad et al. 2010), physiological function of the

The complement system plays an important role in both innate and adaptive host defense against the invading microorganisms in vertebrates. It can be activated by three pathways: the classical, alternative and lectin pathways. Bf/C2 and C4, as members of complement, play a pivotal role in the activation of the complement system. In our study, we identified Bf/C2 and C4 genes and genomic structure in miiuy croaker, and expression patterns of Bf/C2 and C4 genes was analyzed. In healthy miiuy croaker tissues, Bf/C2 and C4 genes were found to be ubiquitously expressed in all ten tested tissues. Analysis of expression of Bf/C2 and C4 genes after bacterial infection showed a significant up-regulated in liver. The evolutionary analysis showed that the ancestral lineages of Bf/C2 and C4 genes in mammals and fishes experienced positive selection indicated that the ancestors of mammals and fishes had further evolved to adapt to their environment, respectively. A series of maximum likelihood (ML) methods were used to study the evolution on vertebrates' Bf/C2 and C4 genes. One and five positive selection sites were found in mammals of Bf/C2 and C4 genes, but no positive selection site was found in fishes of Bf/C2 and C4 genes, indicating that Bf/C2 and C4 genes in mammals and fishes underwent different evolutionary patterns.

Peroxiredoxin family was a superfamily of selenium independent peroxidases. It was divided into six subtypes: Prx1-4 (typical 2-Cys), Prx5 (atypical 2-Cys) and Prx6 (1-Cys). This study reports the isolation and characterization three 2-Cys peroxiredoxin members of full cDNA and genomic clones from miiuy croaker (Miichthys miiuy). The genetic structure analysis showed that the C-terminal catalytic Cys positioned within GEVCPAXW. This sequence was different between Prx3 and Prx4, but was conservative in different species of the same gene, the X base was S in Prx3 but G in Prx4. Tissues expression analysis showed that the expressions of Prx3 in liver and brain were much higher than other tissues; the values of Prx4 in spleen, intestine and kidney were significantly higher than others; and the expression of Prx5 in muscle was higher than that of other tissues. Real-time PCR results showed that there were highest values of these three Prxs emerging with the time post challenge of Vibrio anguillarum in liver, spleen and kidney although the highest value time differed from each other and the expression of these three genes also changed with the change of infection time. These results indicated that expression analysis of these three genes play some positive function against pathogenic bacteria infection in miiuy croaker.

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.

Complement is a humoral factor of innate immunity and plays an essential role in altering the host of the presence of potential pathogens and clearing of invading microorganisms. The third complement component (C3) not only is regarded as the crossing of the three pathways of complement activation, but also serves one of the bridges linking innate and acquired immunity. The nine complement component (C9) can combine with C5b, C6, C7 and C8 to form MAC which bounds to the surface of microorganisms to kill them. The evidence of evolution on C3 genes which have multiple functions and plays central role in innate immunity was documented in our previous study. Now we were interested in the evolution of C9 genes which were the terminal complement components. For these reasons, we want to explore the evolutionary patterns of C9 and whether C3 and C9 experience different evolutionary patterns. In our study, we used the sliding window method to separately calculate the values of ω among fishes and mammals of C3 and C9 codons. In order to detect the positive selection sites, we used the maximum likelihood (ML) method to study the evolutionary pattern on C3 and C9 genes. Positive selection sites were detected in mammalian C9 genes and no positive selection sites were detected in fishes C9 genes. However, no positive selection sites were detected in mammalian C3 genes and positive selection sites were detected in fishes C3 genes. The result indicated that C3 and C9 had different evolutionary patterns on mammals and fishes. In conclusion, different living environments lead to different evolutionary patterns on C3 and C9 in mammals and fishes. Besides, different complement components may have different evolutionary patterns on mammals and fishes.

Myeloid differentiation factor 88 (MyD88) is an important adaptor protein in the TLR signaling pathways. In the present study, we firstly cloned and characterized Miichthys miiuy MyD88 (Mimi-MyD88) cDNA and gene. The Mimi-MyD88 gene was 3,470 bp consisting of five exons and four introns. The cDNA was composed of 1,627 bp with an 867-bp open reading frame encoding a polypeptide of 288 amino acid residues. The theoretical molecular mass and isoelectric point of this polypeptide were 33.25 and 4.96 kDa. Comparison of the deduced amino acid sequence showed that the conserved death domain and the typical Toll/IL-1 receptor domain are very similar to those presented in other mammals, amphibians, and fishes. To identify potential role of MyD88 in fish innate immunological surveillance, the constitutive Mimi-MyD88 mRNA is detected by real-time quantitative polymerase chain reaction. Results demonstrated that Mimi-MyD88 is broadly expressed in ten normal tissues, with the lowest expression was observed in kidney and the highest was in liver. The transcriptional expression also revealed that Mimi-MyD88 was significantly up-regulated in liver, kidney, and spleen after challenge by Gram-negative bacteria, Vibrio anguillarum. Via contrasted the expression of MyD88 and TLR2 in kidney, we evaluated TLR2 plays an indispensable role in MyD88 transcription, but not absolutely dominant. The combined expression still indicated that MyD88 plays a universal role in keeping immune surveillance for pathogens. Phylogenetic analysis suggested that Mimi-MyD88 gene is classified into the piscine cluster and most closely related to large yellow croaker Larimichthys crocea.

Chemokines are a family of structurally related chemotactic cytokines that regulate the migration of leukocytes, under both physiological and inflammatory conditions. A partial cDNA of CC chemokine gene designed as Mimi-CC3 was isolated from miiuy croaker (Miichthys miiuy) spleen cDNA library. Unknown 3' part of the cDNA was amplified by 3'-RACE. The complete cDNA of Mimi-CC3 contains an 89-nt 5'-UTR, a 303-nt open reading frame and a 441-nt 3'-UTR. Three exons and two introns were identified in Mimi-CC3. The deduced Mimi-CC3 protein sequences contain a 22 amino acids signal peptide and a 78 amino acids mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CC chemokines. It shares low amino acid sequence identities with most other fish and mammalian CC chemokines (less than 54.1 %), but shares very high identities with large yellow croaker CC chemokine (94.6 %). Phylogenetic analysis showed that Mimi-CC3 gene may have an orthologous relationship with mammalian/amphibian CCL25 gene. Tissue expression distributed analysis showed that Mimi-CC3 gene was constitutively expressed in all nine tissues examined, although at different levels. Upon stimulated with Vibrio anguillarum, the time-course analysis using a real-time PCR showed that Mimi-CC3 transcript in kidney and liver was obviously up-regulated and reached the peak levels, followed by a recovery. Mimi-CC3 expression in kidney was more strongly increased than in liver. However, down-regulation was observed in spleen. These results indicated that Mimi-CC3 plays important roles in miiuy croaker immune response as well as in homeostatic mechanisms.

The laboratory of genetics and physiology 2 (LGP2) is a member of retinoic acid-inducible gene I (RIG-I)-like receptors (RLR receptors), which may participate in the immune regulation process. The role of LGP2 on modulating signaling was ambiguous, some researchers suggested that the regulation mechanism of LGP2 to melanoma differentiation-associated gene 5 (MDA5) and retinoic acid inducible gene-I (RIG-I) were different. In this study, the bioinformatics and functions of LGP2 from miiuy croaker (mmLGP2) were characterized. Comparative genomic analysis showed that the evolution of LGP2 in mammals was more conserved than it in fish. LGP2 contains three structural domains: ResIII, HelicaseC and RD, and ResIII structural domain of LGP2 was extremely conservative. The mmLGP2 was ubiquitously expressed in the tested miiuy croaker tissues and the expressions were significantly upregulated after stimulation with poly(I:C), indicating that LGP2 might participate in the immune response, especially antiviral immunity. Furthermore, immunofluorescence of miiuy croaker LGP2 presents in the cytoplasm in Hela cells. The overexpression of mmLGP2 can activate ISRE, but cannot activate NF-κB luciferase reporter, implying that mmLGP2 might act as a positive regulator in immune responses through activating ISRE to induce the expression of IFN. The research of mmLGP2 will enrich the information of fish LGP2, and the functional experiments will be helpful for the future research about fish immune systems.

IFN regulatory factor (IRF) 3 as an important member of IRF family, is required for the host antiviral response. In mammals, IRF3 is known to be a critical player in regulating the transcription of IFN and IFN-stimulated genes (ISGs). However, only a few studies investigated the characteristics of IRF3 genes in fish. In this study, IRF3 from miiuy croaker was identified and characterized in bioinformatics and functions. Miiuy croaker IRF3 had conserved DBD, IAD and SRD domains with other vertebrates IRF3 genes, also miiuy croaker IRF3 had relatively conserved gene synteny and gene structures with other fish IRF3 genes. Evolutionary analysis showed IRF3 genes in mammals underwent positive selection, while IRF3 in fish underwent purifying selection. Expression analysis showed miiuy croaker IRF3 was expressed in all tested tissues and up-regulated expressed in infected liver and kidney; and up-regulated expression of miiuy croaker IRF3 was observed in head kidney macrophages which stimulated with poly(I:C) indicating that miiuy croaker IRF3 participated in the immune response to defense against poly(I:C) infection. Furthermore, luciferase reporter assay showed that overexpression of miiuy croaker IRF3 can activate the production of ISRE and IFNα, suggesting that miiuy croaker IRF3 acted as transcription activators in immune responses and maybe activate IFN signaling pathway. Immunofluorescence assay showed miiuy craoker IRF3 was localized in the cytoplasm in Hela cells. Overall, we systematically and comprehensively analyzed the bioinformatics and functions of miiuy croaker IRF3, which provided further insights into the transcriptional regulation of IRF3 gene in fish and valuable information for the study of evolution of IRF3 genes.

MicroRNAs (miRNAs) as endogenous small non-coding RNAs play key regulatory roles in diverse biological processes via degrading the target mRNAs or inhibiting protein translation. Previously many researchers have reported the identification, characteristic of miRNAs and the interaction with its target gene. But, the study on the regulation of miRNAs to biological processes via regulatory the key signaling pathway was still limited. In order to comprehend the regulatory mechanism of miRNAs, two small RNA libraries from the spleen of miiuy croaker individuals with or without poly(I:C) infection were constructed. The 197 conserved miRNAs and 75 novel miRNAs were identified, and 14 conserved and 8 novel miRNAs appeared significant variations. Those differently expressed miRNAs relate to immune regulation of miiuy croaker. Furthermore, expressions of four differently expressed miRNAs were validated by qRT-PCR, and the result was consistent with sequencing data. The target genes of the differently expressed miRNAs in the two libraries were predicted, and some candidate target genes were involved in the RIG-I-like receptor (RLR) signaling pathway. The negative regulation of miRNAs to target genes were confirmed by comparing the expression pattern of miRNAs and their target genes. The results of regulating target genes were that firstly directly or indirectly activating the downstream signaling cascades and subsequent inducting the type I interferon, inflammatory cytokines and apoptosis. These studies could help us to deeper understand the roles of miRNAs played in the fish immune system, and provide a new way to investigate the defense mechanism of fish.

Activation of innate and acquired immune responses is regulated by detailed mechanisms to control their onset and termination. MicroRNAs have been implicated as negative regulators controlling the diverse of biophysical and biochemical processes at the post-transcriptional level. However, the physiological roles of miRNAs in aquatic organisms are largely unclear. In this study, we explored the potential roles of mmi-miR-192 in regulating interleukin 1 receptor type I (IL-1RI) involved in immune and inflammatory response in miiuy croakers. This was further evaluated by negative expression profiles in both LPS exposure macrophages and Vibrio anguillarum challenged miiuy croaker. By means of promoter analysis, mmi-miR-192 was found to be an AP-1 dependent gene. Importantly, the dual luciferase reporter assay presented the regulation between mmi-miR-192 and IL-1RI. The result of miiuy croaker miR-192 reduced the wild-type IL-1RI but not the mutant one luciferase levels suggested that mmi-miR-192 modulated IL-1RI expression by directly targeting the 3'UTR of IL-1RI mRNA. Overall, our study revealed the mechanism that the miR-192-IL1RI pathway regulated bacteria infection in miiuy croakers.

Major histocompatibility complex (MHC) molecules play a vital role in the immune response and are a highly polymorphic gene superfamily in vertebrates. As the molecular marker associated with polymorphism and disease susceptibility/resistance, the polymorphism of MHC genes has been investigated in many tetrapods and teleosts. Most studies were focused on the polymorphism of the second exon, which encodes the peptide-binding region (PBR) in the α1- or β1-domain, but few studies have examined the full-length coding region. To comprehensive investigate the polymorphism of MHC gene, we identified 48 full-length miiuy croaker (Miichthys miiuy) MHC class IIB (Mimi-DAB) functional alleles from 26 miiuy croaker individuals. All of the alleles encode 34 amino acid sequences, and a high level of polymorphism was detected in Mimi-DAB alleles. The rate of non-synonymous substitutions (dN) occurred at a significantly higher frequency than that of synonymous substitutions (dS) in the PBR, and this result suggests that balancing selection maintains polymorphisms at the Mimi-DAB locus. Phylogenetic analysis based on the full-length and exon 2 sequences of Mimi-DAB alleles both showed that the Mimi-DAB alleles were clustered into two major groups. A total of 19 positive selected sites were identified on the Mimi-DAB alleles after testing for positive selection, and 14 sites were predicted to be associated with antigen-binding sites, which suggests that most of selected sites are significant for disease resistance. The polymorphism of Mimi-DAB alleles provides an important resource for analyzing the association between the polymorphism of MHC gene and disease susceptibility/resistance, and for researching the molecular selective breeding of miiuy croaker with enhanced disease resistance.

The golden drum (Chrysochir aurenus) is an important food fish of Indo-West Pacific and China. To study the phylogenetic status, we sequenced the complete mitochondrial genome of C. aurenus. The mitogenome is 16 505 bp in length and composed of 13 protein-coding genes, 2 rRNAs, 22 tRNAs, and a control region. The gene composition and the structural arrangement of C. aurenus complete mtDNA were identical to most of other vertebrates. The phylogenetic analysis using the complete mitochondrial genome revealed that the C. aurenus might be grouped in different genera of Argyrosominae, but not belonged to Otolithinae, which was highly consistent with that based on the morphology. The present study will be helpful for the evolution and conservation genetic studies of C. aurenus.

Chemokines control leukocyte trafficking which plays important roles in resistance to pathogenic infection. Here, a full-length CC chemokine (MimiCC2) cDNA was isolated from miiuy croaker ( Miichthys miiuy ) by EST analysis and RACE-PCR. Its open reading frame is 300 nucleotides, encoding a polypeptide of 99 amino acids. The deduced MimiCC2 contains a 23-aa signal peptide and a 76-aa matural peptide, which possesses the four invariant cysteine residues as found in other known CC chemokines. It shares 13.3% to 73.7% sequence identities to mammalian and fish CC chemokines, with the highest identity observed between MimiCC2 and seabream CCL4. Homology modeling showed that MimiCC2 had a tertiary structure containing three β-strands and an α-helix. Three exons and two introns were identified in miiuy croaker CC. Phylogenetic analysis showed that miiuy croaker CC is similar to CCL4. In normal miiuy croaker, MimiCC2 was expressed in all the tissues examined. Infection of miiuy croaker with Vibrio anguillarum significantly increased expression of MimiCC2 in the immune-related organs. These results indicated that MimiCC2 probably plays important role in miiuy croaker immune system.

The complete mitochondrial genome sequence of the marbled rockfish Sebastiscus marmoratus (Scorpaeniformes, Scorpaenidae) was determined and phylogenetic analysis was conducted to elucidate the evolutionary relationship of the marbled rockfish with other Sebastinae species. This mitochondrial genome, consisting of 17301 bp, is highly similar to that of most other vertebrates, containing the same gene order and an identical number of genes or regions, including 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and one putative control region. Most of the genes are encoded on the H-strand, while the ND6 and seven tRNA genes (for Gln, Ala, Asn, Tyr, Ser (UCA), Glu, and Pro) are encoded on the L-strand. The reading frame of two pairs of genes overlapped on the same strand (the ATPase 8 and 6 genes overlapped by ten nucleotides; ND4L and ND4 genes overlapped by seven nucleotides). The possibly nonfunctional light-strand replication origin folded into a typical stem-loop secondary structure and a conserved motif (5′-GCCGG-3′) was found at the base of the stem within the tRNACys gene. An extent termination-associated sequence (ETAS) and conserved sequence blocks (CSB) were identified in the control region, except for CSB-1; unusual long tandem repeats were found at the 3′ end of the control region. Phylogenetic analyses supported the view that Sebastinae comprises four genera (Sebastes, Hozukius, Helicolenus, and Sebastiscus).

Cathepsin S (CTSS) is a lysosomal cysteine endopeptides of the papain family. In the present study, CTSS gene was isolated from miiuy croaker (Miichthys miiuy) by expressed sequence tag analysis. The complete miiuy croaker CTSS cDNA comprised 1,454 nucleotides, including 56 bp at the 5'-UTR and 394 bp at the 3'-UTR. The open reading frame is 1,017 bp, and it encodes 338 amino acid residues with a calculated molecular weight of 37.1 kDa. BLAST analysis revealed that miiuy croaker cathepsin S shared high similarity with other known cathepsin S, and it showed significant homology with that of Japanese flounder, the degree of conservation of the miiuy croaker CTSS nucleotide sequence in comparison with other teleost species ranged from 63.0 to 81.7%. The N-linked glycosylation sites and catalytic sites are conserved in miiuy croaker CTSS. The CTSS transcript was expressed in all tissues examined; high levels of transcripts of CTSS were detected liver, muscle, and fin. These results provide important information for further exploring the roles of cathepsin S in antigen processing.

Major histocompatibility complex (MHC) genes play an important role in the immune response of vertebrates. Allelic polymorphism and evolutionary mechanism of MHC genes have been investigated in many mammals, but much less is known in teleosts. We examined the polymorphism, gene duplication and balancing selection of the MHC class II DAB gene of the half-smooth tongue sole (Cynoglossus semilaevis); 23 alleles were found in this species. Gene duplication manifested as three to six distinct sequences at each domain in the same individuals. Non-synonymous substitutions occurred at a significantly higher frequency than synonymous substitutions in the PBR domain, suggesting balancing selection for maintaining polymorphisms at the MHC II DAB locus. Many positive selection sites were found to act very intensely on antigen-binding sites of MHC class II DAB gene.

In this study, the complete mitochondrial genome of yellow goosefish Lophius litulon (L. litulon) was determined. The genome was 16,431 bp in length, and comprised 13 protein-coding genes, 2 ribosomal RNA gene and 22 transfer RNA genes, with 2 main non-coding regions. Its gene order and composition was similar to most of the vertebrates. The overall base composition of L. litulon was 28.6% A; 29.1% C; 16.8% G; 25.5% T, with a slight AT bias of 54.1%. The origin of L-strand replication in L. litulon was located in a cluster of five tRNA genes (WANCY). Motif TCCGG was found in the origin of light strand replication (OL) of L. litulon, which was common in other mitogenome of Lophiidae. This mitogenome data would play key role in future studying phylogenetic analysis of the Lophiidae.

Allelic polymorphism and evolution mechanism of major histocompatibility complex (MHC) genes has been investigated in many mammals, however, much less is known in teleost. In order to investigate the mechanisms creating and maintaining variability at the MHC class II DAA locus, we examined the polymorphism, gene duplication and balancing selection of MHC class II DAA gene of the half-smooth tongue sole (Cynoglossus semilaevis). We described 33 alleles in the C. semilaevis, recombination and gene duplication seems to play more important roles in the origin of new alleles. The rate of non-synonymous substitutions (d(N)) occurred at a significantly higher frequency than that of synonymous substitutions (d(S)) in peptide-binding region (PBR) and non-PBR, suggesting balancing selection for maintaining polymorphisms at the MHC II DAA locus. Many positive selection sites were found to act very intensively on antigen-binding sites. Our founding suggests a snapshot in an evolutionary process of MHC-DAA gene evolution of the C. semilaevis.

Interleukin-17 (IL-17) family is a cytokine family which is one of the major signaling molecules family involved in immunity. Six member of IL-17 family cytokines (IL-17A-F) were found in mammals. In fish, all IL-17 family genes except IL-17B and IL-17E have been isolated and identified. Besides, IL-17N is uniquely found from teleosts. IL-17 family genes are widely studied in mammals, but have not been widely reported in lower vertebrates. In this study, we identify six IL-17 family genes (IL-17A/F1-3, IL-17C, IL-17D, IL-17N) from miiuy croaker, using LPS and poly (I:C) to infect miiuy croaker in order to analyze the expression response to bacteria and virus and expression in normal tissues. Challenge experiment showed that miiuy croaker IL-17 family genes exhibited more sensitive response to the poly (I:C) than the LPS. The expression of IL-17 in un-stimulated tissues showed that different gene has expressed in different tissues. Through the analysis of IL-17 family members exist in various representative species to study the evolution of the IL-17 family, and the result showed IL-17A/F, IL-17B, IL-17C, and IL-17D should be present in early gnathostomes species.

The interferon regulatory factors IRF1 and IRF2 of the IRF1 subfamily play essential roles in immune responses against viruses. IRF11 is a novel IRF gene of the IRF1 subfamily; IRF11 genes share almost the same evolutionary distance with IRF1 and IRF2 genes. However, the structure and characteristics of IRF11 gene in fish have been rarely reported. In our study, IRF1, IRF2 and IRF11 genes were identified and characterized from miiuy croaker genome. Results showed that the IRF1, IRF2 and IRF11 genes contain the same domains; each of these genes is composed of conserved gene organizations and characterized by gene synteny with the orthologous genes. Interestingly, IRF11 was likely found only in fish (but not specific to teleost fish). Evolutionary analysis results showed that IRF1 gene in mammals, IRF2 and IRF11 gene in fish underwent positive selection. IRF1, IRF2 and IRF11 were expressed in a wide range of miiuy croaker tissues. These genes also exhibited the same expression patterns after miiuy croaker was infected with poly(I:C). Therefore, our data enhanced our understanding of the functions and evolution of IRF11 in fish.

The nucleotide-binding oligomerization domain proteins NOD1 and NOD2 are important cytoplasmic pathogen recognition receptors which sense microbial infections molecules to induce innate immune response. In this study, the sequence analysis showed that NOD1 and NOD2 genes in miiuy croaker (miichthys miiuy, mmiNOD1 and mmiNOD2) share some highly conserved motifs that crucial for recognizing the bacterial and viral components. Quantitative expression analysis revealed mmiNOD1 and mmiNOD2 had the highest level of expression in liver. Induction experiments with Vibrio anguillarum indicated the different expression levels of mmiNOD1 and mmiNOD2 in liver, spleen and kidney. The expressions of mmiNOD1 and mmiNOD2 increased more significantly after Poly(I:C) stimulation, meanwhile, we carried out the expression analysis at the transcriptome level and the regulation of microRNAs. In addition, the evolutionary analysis showed that the ancestral lineages of NOD1 in bony fish detected one positively selected site, however, both the current lineages of NOD1 and NOD2 genes in bony fish underwent purifying selection indicating that NOD1 gene in the ancestor of bony fish experienced positive selection. To further understand the evolutionary pattern of NOD1 and NOD2 in vertebrates, we were the first to conduct comparative genomic analysis by comparing the number and synteny of NOD1 and NOD2. Combining the duplication of NOD1, the lost of NOD2 and the more conserved synteny of NOD2 than NOD1, we proposed that the hypothetical evolutionary pattern is different between NOD1 and NOD2.

Bombay duck, Harpodon nehereus (Aulopiformes, Synodontidae), is a commercially important marine fish species in China. In this study, the complete mitochondrial genome of H. nehereus was firstly determined. The mitogenome (16 and 536 bp) comprises 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 putative control region. These mitogenome sequence data would play an important role in population genetics and phylogenetic analysis of Aulopiformes.

The Asian freshwater goby, Synechogobius ommaturus (Perciformes, Gobiidae) is a big goby inhabiting shallow sandy-muddy bottoms in temperate riverine estuaries and inner bays, mainly distributed in China, Japan, and the Korean Peninsula. In this paper, the complete mitochondrial genome of S. ommaturus was firstly determined. The genome is 16,660 bp in length and consists of 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, and 2 main non-coding regions (the control region and the origin of the light strand replication). The overall base composition of S. ommaturus is 26.9% for T, 27.9% for C, 27.7% for A, and 17.4% for G, with a slight A+T bias of 54.6%. It has the typical vertebrate mitochondrial gene arrangement.

The Walking goby Scartelaos histophorus (Perciformes, Gobiidae) is an amphibious gobioid fish. In this paper, the complete mitochondrial genome of S. histophorus was first determined. The genome is 16,496 bp in length and consists of 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, and 1 control region. The overall base composition of S. histophorus is 27.5% for T, 28.0% for C, 28.3% for A, and 16.1% for G, with a slight A+T bias of 55.8%. It has the typical vertebrate mitochondrial gene arrangement.

The complete mitochondrial genome (mitogenome) of Oxuderces dentatus was determined first. The genome was 17,116 bp in length and consisted of 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, and 2 main non-coding regions [the control region (CR) and the origin of the light strand replication], the gene composition and order of which was similar to most other vertebrates. The overall base composition of the heavy strand was T 27.9%, C 26.8%, A 30.2%, and G 15.1%, with a slight A+T bias of 58.1%. In addition to the discrete and conserved sequence blocks, unusual long tandem repeat unit (three 150-bp tandem repeat units and an incomplete copy of 146 bp) was also detected within CR. This mitogenome sequence data would play an important role in population genetics and phylogenetic analysis of the Gobioidei.

Highlights • Miiuy croaker DC-SIGN and L-SIGN have been comprehensively analyzed. • An additional L-SIGN-like gene exists in some fishes which may be from L-SIGN duplication. • We are the first to explore conserved gene synteny of L-SIGN in teleosts. • The current lineages of L-SIGN in fishes undergo positive selection. • Miiuy croaker DC-SIGN and L-SIGN play important roles in immune response., Dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN/CD209) and liver/lymph node-specific ICAM-grabbing non-integrin (L-SIGN/CD299) which are homologues of DC-SIGN are important members in C-type lectin receptors family as key molecules to recognize and eliminate pathogens in the innate immune system. DC-SIGN and L-SIGN have become hot topics in recent studies which both served as cell adhesion and phagocytic pathogen recognition receptors in mammals. However, there have been almost no studies of DC-SIGN and L-SIGN structure and characters in fish, only DC-SIGN in the zebrafish had been studied. In our study, we identified and characterized the full-length miiuy croaker (Miichthys miiuy) DC-SIGN (mmDC-SIGN) and L-SIGN (mmL-SIGN) genes. The sequence analysis results showed that mmDC-SIGN and mmL-SIGN have the same domains with other vertebrates except primates, and share some conserved motifs in CRD among all the vertebrates which play a crucial role in interacting with Ca2+ and for recognizing mannose-containing motifs. Gene synteny of DC-SIGN and L-SIGN were analyzed for the first time and gene synteny of L-SIGN was conserved among the five fishes. Interestingly, one gene next to L-SIGN from gene synteny had high similarity with L-SIGN gene that was described as L-SIGN-like in fish species. While only one L-SIGN gene existed in other vertebrates, two L-SIGN in fish may be in consequence of the fish-specific genome duplication to adapt the specific environment. The evolutionary analysis showed that the ancestral lineages of L-SIGN gene in fishes experienced purifying selection and the current lineages of L-SIGN gene in fishes underwent positive selection, indicating that the ancestral lineages and current lineages of L-SIGN gene in fishes underwent different evolutionary patterns. Both mmDC-SIGN and mmL-SIGN were expressed in all tested tissues and ubiquitously up-regulated in infected liver, spleen and kidney at different sampling time points, indicating that the mmDC-SIGN and mmL-SIGN participated in the immune response to defense against bacteria infection.

As a member of the tumor necrosis factor (TNF) family, B cell activating factor (BAFF), also known as TNF ligand superfamily member 13B (TNF13B), playing a critical role in enhancing immune responses. BAFF is a central cytokine for B-cell survival, proliferation, maturation and immunoglobulin secretion. In the present study, we describe the identification of the miiuy croaker BAFF (designed MmBAFF) and BAFF-like (designed MmBAFF-like) genes. The cDNA of MmBAFF contains an open reading frame (ORF) of 795 nucleotides that are translated into a predicted 264 amino acids. The ORF of MmBAFF-like consists of 705 bases encoding 234 amino acids. Amino acid sequence comparison indicated that MmBAFF and MmBAFF-like possessed the TNF signatures, a predicted transmembrane domain, three conserved cysteine residues and a putative furin protease cleavage site, which were the typical characteristics of TNF gene in mammals and birds. The predicted three-dimensional (3D) structure of the MmBAFF and MmBAFF-like monomer analyzed by comparative protein modeling revealed that they were very similar to human counterpart. Comparative genomic analysis revealed that the locations of MmBAFF and MmBAFF-like genes are conserved among the bony fish. Phylogenetic analysis shows the MmBAFF is most closely related to other teleost BAFFs with the highest similarity to Epinephelus awoara. And BAFF-like cluster get together first to BAFF cluster than three closely related TNF superfamily (TNFSF) members. Real-time quantitative PCR analysis shows the MmBAFF and MmBAFF-like transcripts are expressed in a wide range of tissues with the highest expression in skin and lymphoid tissue spleen. Upon induction by Vibrio anguillarum, their expressions are significantly upregulated in liver, spleen and kidney as compared to phosphate-buffered saline injected control fish. The association of increased BAFF expression after bacterial infection suggests that it plays a potentially important role in immune system of fish.