Yuena Sun

Identification of immune genes of the miiuy croaker (Miichthys miiuy) by sequencing and bioinformatic analysis of ESTs.

Miiuy croaker (Miichthys miiuy) is an economically important fish in China. However, genomic research on this species is still in its infancy, and genomic resources are largely unavailable. In order to isolate functional genes involved in immunity, a normalized cDNA library was constructed from the spleen of the miiuy croaker. A total of 5053 ESTs from the library were sequenced and compared with sequences in the GenBank database. The 4609 high-quality ESTs were assembled into 3221 unigenes. Based on sequence similarities, 193 immune genes were identified such as major histocompatibility complex, cytokines and cytokine receptors, adhesive proteins, stress proteins, transcription factors for immune response, immunoglobulin and coagulation factors. Our study thus provides both a detailed annotation of immune genes in miiuy croaker and a collection of novel transcripts of Fc receptor-like 5 in teleost for the first time.

The genome of the miiuy croaker reveals well-developed innate immune and sensory systems

The miiuy croaker, Miichthys miiuy, is a representative Sciaenidae known for its exceptionally large otoliths. This species mainly inhabits turbid aquatic environments with mud to sandy mud bottoms. However, the characteristics of the immune system of this organism and its specific aquatic environment adaptations are poorly understood. Thus, we present a high-quality draft genome of miiuy croaker. The expansions of several gene families which are critical for the fish innate immune system were identified. Compared with the genomes of other fishes, some changes have occurred in the miiuy croaker sensory system including modification of vision and expansion of taste and olfaction receptors. These changes allow miiuy croaker to adapt to the environment during the long-term natural selection. The genome of miiuy croaker may elucidate its relatively well-developed immune defense and provide an adaptation model of the species thriving in turbid deep aquatic environments.

Characterization of the Major Histocompatibility Complex Class II Genes in Miiuy Croaker

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.

Miiuy Croaker Hepcidin Gene and Comparative Analyses Reveal Evidence for Positive Selection

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.

Transcriptomic analysis of endangered Chinese salamander: identification of immune, sex and reproduction-related genes and genetic markers.

Background 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. Principal Findings 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). Conclusion 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.

Molecular characterization of miiuy croaker CC chemokine gene and its expression following Vibrio anguillarum injection

A CC chemokine gene was isolated from miiuy croaker (Miichthys miiuy) by expressed sequence tag analysis. The Mimi-CC cDNA contains an open reading frame of 429 nucleotides encoding 142 amino acid residues. The deduced Mimi-CC possesses the typical arrangement of four cysteines as found in other known CC chemokines (C³¹, C³², C⁵⁶, and C⁷⁰). It shares 15.3%-37.4% identity to CC chemokines of mammal and teleost. Phylogenetic analysis showed that miiuy croaker was most closely related to Atlantic cod. Genomic analysis revealed that Mimi-CC gene consists of four exons and three introns, which is not typical of CC chemokines but resembles that of CXC chemokines. Real-time quantitative RT-PCR demonstrated that Mimi-CC is constitutively expressed in most tissues including lymphoid organs, and the highest expression of Mimi-CC transcripts in normal tissues was observed in muscle. Challenge of miiuy croaker with Vibrio anguillarum resulted in significant changes in the expression of CC chemokine transcripts in four tissues, especially in kidney and spleen.

Characterization and expression of the CXCR1 and CXCR4 in miiuy croaker and evolutionary analysis shows the strong positive selection pressures imposed in mammal CXCR1

The innate immune system can recognize non-self, danger signals, and pathogen associated molecular patterns and provides a first line of antimicrobial host defense. Therefore, it plays an instructive role and is pretty important in vertebrates. In innate immune responses, CXCRs act as the main receptors of CXC chemokines and play a vital role in host defense and inflammation. In present study, we cloned two cDNA molecules of CXCR1 and CXCR4 in Miichthys miiuy (miiuy croaker). In these two genes, we found the most highly conserved DRY motif in the second intracellular loop adjacent to the third transmembrane domain. The expressions of CXCR1 and CXCR4 showed that they were ubiquitously expressed in ten normal tissues. After infection with Vibrio anguillarum and Vibrio harveyi, the expressions of CXCRs in the immune tissues were significantly regulated in most of tissues except that of CXCR1 in the kidney after V. harveyi injection. Evolutionary analysis showed that only the ancestral lineages of CXCR4 in amphibians underwent positive selection, indicating that the ancestors of amphibians boarded the land and had to further evolve to adapt to terrestrial environments. Multiple ML methods were implemented to detect the robust positively selected candidates for sites. In total, we detected 12 and 3 positively selected sites in the subsets of current mammal and fish CXCR1 genes, and only one site under positive selection was found in mammalian CXCR4 subsets. These positively selected sites were mainly located in the extracellular domains of CXCRs. The sliding window analysis and evolution test tended to favor positive selection acting on the N-terminal domain of CXCR1, which was the critical region for ligand/receptor signaling for neutrophils and receptor-ligand interaction, indicating that the N-terminal of CXCR1 in mammals underwent more positive selection than that of fish.

Miiuy Croaker Transferrin Gene and Evidence for Positive Selection Events Reveal Different Evolutionary Patterns

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.

The complete mitochondrial genome of the small yellow croaker and partitioned Bayesian analysis of Sciaenidae fish phylogeny

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.

Genomic sequences comparison and differential expression of miiuy croaker MHC class I gene, after infection by Vibrio anguillarum

Major histocompatibility complex (MHC) has a central role in the adaptive immune system by presenting foreign peptide to the T-cell receptor. MHC gene family contains two main subgroups of immunologically active molecules. In order to study the molecular function and genomic characteristic of class I gene in teleost, the full lengths of MHC class Iα cDNA and genomic sequence were cloned from miiuy croaker (Miichthys miiuy). Seven exons and six introns were identified in miiuy croaker class Iα gene. This genomic structural feature of miiuy croaker is similar to that present in some fishes such as Japanese flounder and Atlantic salmon, but different from that present in some other fishes such as half-smooth tongue sole and channel catfish. The deduced amino acid sequence of class Iα gene had 25.9-54.1% identity with those of mammal and teleost. Real-time quantitative RT-PCR demonstrated that the MHC class Iα gene was ubiquitously expressed in 10 normal tissues; expression levels of MHC Iα gene were found first upregulated and then downregulated throughout the pathogenic bacteria infection process in spleen and kidney.