Characterization of a novel antimicrobial peptide with chitin-biding domain from Mytilus coruscus.

Using reverse phase high performance liquid chromatography (RP-HPLC), a novel antimicrobial peptide with 55 amino acid residues was isolated from the hemolymph of Mytilus coruscus. This new antimicrobial peptide displays predominant antimicrobial activity against fungi and Gram-positive bacteria. The molecular mass and the N-terminal sequence of this peptide were analyzed by Mass Spectrometry and Edman degradation, respectively. This antimicrobial peptide, with molecular mass of 6621.55 Da, is characterized by a chitin-biding domain and by 6 Cysteine residues engaged in three intra-molecular disulfide bridges. The full-length of cDNA sequence of this new peptide was obtained by rapid amplification of cDNA ends (RACE) and the encoded precursor was turn out to be a chitotriosidase-like protein. Therefore, we named the precursor with mytichitin-1 and the new antimicrobial peptide (designated as mytichitin-CB) is the carboxyl-terminal part of mytichitin-1. The mRNA transcripts of mytichitin-1 are mainly detected in gonad and the expression level of mytichitin-1 in gonad was up-regulated and reached the highest level at 12 h after bacterial challenge, which was 9-fold increase compared to that of the control group. These results indicated that mytichitin-1 was involved in the host immune response against bacterial infection and might contribute to the clearance of invading bacteria.

[Characteristics and phylogenetic analysis of mitochondrial genome in the gobies].

The vast number of species, small size and high variation of morphology make the morphological identification and classification of gobies very difficult. In this study, the complete mitochondrial genome (mitogenome) of 26 species of gobies was analyzed, aiming at accumulating the molecular information on the identification, classification and molecular evolution of gobies. The results showed that the gene composition and arrangement of mitogenome of gobies are similar to most vertebrates. Due to various degrees of repetitive sequences in the control region, the mitogenome of 26 gobies exhibits a great variation in length. The A+T content of the mitogenome is greater than 50% and the lowest frequency is for G among the four bases. Thirty-seven coding gene sequences were used to calculate the average Kimura 2-parameter genetic distance of 26 species of gobies. Acanthogobius hasta and A. ommaturus, Glossogobius olivaceus and G circumspectus were synonyms, respectively. By comparing the control region sequences of 26 gobies, the terminal associated sequences, central conserved sequence block and conserved sequence block were identified, respectively. Thirty-six coding gene sequences of 26 gobies were used to construct the phylogenetic tree and the results were different from the traditional morphological classification. The five subfamilies of Gobiidae were obviously evolved: Amblyopinae, Oxudercinae and Sicydiinae were clustered into a group and then formed a sister group with Gobionellinae; the fishes of Gobiinae had distant relationship with the four subfamilies and formed a group alone. Molecular clock analysis estimated that gobies probably originated in the late Eocene to Oligocene time and further evolved into modern characteristic gobies in the Miocene.

Molecular cloning, characterization and expression analysis of a miiuy croaker (Miichthys miiuy) CXC chemokine gene resembling the CXCL9/CXCL10/CXCL11.

Chemokines are a large family of chemotactic cytokines playing crucial roles in the innate immune response. In the present study, we report the cloning of a CXC chemokine gene resembling the closely related CXCL9/CXCL10/CXCL11 from the miiuy croaker Miichthys miiuy (MimiCXC). Both 5'-RACE and 3'-RACE were carried out in order to obtain the complete cDNA, which consists of a 73 bp 5'-UTR, a 369 bp open reading frame encoding 122 amino acids and a 715 bp 3'-UTR. The deduced MimiCXC contains a 19-aa signal peptide and a 103-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CXC chemokines. It shares 4.8%-65.6% sequence identities to mammalian CXC chemokines and the highest sequence identity of 65.6% is between MimiCXC and CXCL10 chemokine. Three exons and two introns were identified in MimiCXC gene. The MimiCXC gene was constitutively expressed in all tissues tested, although at different levels. Upon induction with Vibrio anguillarum, MimiCXC gene expression was up-regulated in kidney and spleen, however, down-regulated in liver. These results indicate that MimiCXC may be involved in immune responses as well as homeostatic processes in miiuy croaker.

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 C7°). 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.

The complete mitochondrial genome of bighead croaker, Collichthys niveatus (Perciformes, Sciaenidae): structure of control region and phylogenetic considerations.

Sciaenidae is a diverse, commercially important family. To understand the phylogenetic position of Collichthys niveatus in this family, we present its complete mitochondrial genome sequence. The genome is 16469 bp in length and contains 37 mitochondrial genes (13 protein-coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes) and a control region (CR) as in other bony fishes. Further sequencing for the complete control region was performed on Collichthys lucida. Although the conserved sequence domains such as extend termination associated sequence (ETAS) and conserved sequence block domains (CSB-1, CSB-2 and CSB-3) are recognized in the control region of the two congeneric species, the typical central conserved blocks (CSB-F, CSB-E and CSB-D) could not be detected, while they are found in Miichthys miiuy and Cynoscion acoupa of Sciaenidae and other Percoidei fishes. Phylogenetic analyses do not support the monophyly of Pseudosciaeniae, which is against with the morphological results. C. niveatus is most closely related to Larimichthys polyactis, and Collichthys and Larimichthys may be merged into one genus, based on the current datasets.

Sequence and expression analysis of cathepsin S gene in the miiuy croaker Miichthys miiuy.

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.

The complete mitochondrial genome of the marbled rockfish Sebastiscus marmoratus (Scorpaeniformes, Scorpaenidae): genome characterization and phylogenetic considerations.

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 tRNA(Cys) 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 (Sebates, Hozukius, Helicolenus, and Sebasticus).

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.

[Succession Pattern of Phytoplankton and Its Drivers in Lake Luoma, Jiangsu Province].

As a water storage lake for the South-to-North Water Diversion Project, it is crucial to examine changes in aquatic ecosystem structures in Lake Luoma, Jiangsu province. Field sampling was carried out in Lake Luoma monthly from 2014 to 2018 to study the relationship between the phytoplankton community structure and environmental factors. During the studied period, total nitrogen, permanganate index, and electrical conductivity in water column gradually increased, whereas fluoride content declined. The pattern of total phosphorus and dissolved oxygen was not distinct. A total of 71 genera of phytoplankton were identified from 2014 to 2018, and the average monthly biomass variation ranged from 0.16 to 5.51 mg·L-1. Bacillariophyta and Chlorophyta were the dominant phyla in the four years, followed by Pyrrophyta and Cryptophyta. The dominant genera were Synedra sp., Chroomonas spp., Aulacoseira spp., Dinobryon sp., Scenedesmus spp. , Fragilaria spp., Mougeotia sp. , Ankistrodesmus sp. , and Euglena spp. The results showed that the phytoplankton community structure significantly changed in the four years, which was mainly ascribed to the redistribution of biomass. Specifically, in addition to the dominance of Bacillariophyta and Chlorophyta, the dominance of Pyrrophyta and Cyanophyta increased during the last two years. Non-metric multidimensional scaling analysis showed that variation of the phytoplankton community in Lake Luoma was mainly related to total nitrogen, fluoride, water temperature, total phosphorus, dissolved oxygen, pH, conductivity, and permanganate index, among which the total nitrogen, water temperature, and fluoride concentration dominated the phytoplankton community change after the generalized additive model test. Water temperature is the driving factor affecting seasonal changes of the phytoplankton community. Total nitrogen and fluoride concentrations are the driving factors affecting the interannual variation in the phytoplankton community. Our study indicated that in recent years, the implementation of the ban on sand mining and demolition of the enclosed aquaculture in Lake Luoma has affected the water environment, resulting in a significant succession of the phytoplankton community.

Excess melanin precursors rescue defective cuticular traits in stony mutant silkworms probably by upregulating four genes encoding RR1-type larval cuticular proteins.

Melanin and cuticular proteins are vital cuticle components in insects. Cuticular defects caused by mutations in cuticular protein-encoding genes can obstruct melanin deposition. The effects of changes in melanin on the expression of cuticular protein-encoding genes, the cuticular and morphological traits, and the origins of these effects are unknown. We found that the cuticular physical characteristics and the expression patterns of larval cuticular protein-encoding genes markedly differed between the melanic and non-melanic integument regions. By using four p multiple-allele color pattern mutants with increasing degrees of melanism (+p, pM, pS, and pB), we found that the degree of melanism and the expression of four RR1-type larval cuticular protein-encoding genes (BmCPR2, BmLcp18, BmLcp22, and BmLcp30) were positively correlated. By modulating the content of melanin precursors and the expression of cuticular protein-encoding genes in cells in tissues and in vivo, we showed that this positive correlation was due to the induction of melanin precursors. More importantly, the melanism trait introduced into the BmCPR2 deletion strain Dazao-stony induced up-regulation of three other similar chitin-binding characteristic larval cuticular protein-encoding genes, thus rescuing the cuticular, morphological and adaptability defects of the Dazao-stony strain. This rescue ability increased with increasing melanism levels. This is the first study reporting the induction of cuticular protein-encoding genes by melanin and the biological importance of this induction in affecting the physiological characteristics of the cuticle.

Mutation of a cuticular protein, BmorCPR2, alters larval body shape and adaptability in silkworm, Bombyx mori.

Cuticular proteins (CPs) are crucial components of the insect cuticle. Although numerous genes encoding cuticular proteins have been identified in known insect genomes to date, their functions in maintaining insect body shape and adaptability remain largely unknown. In the current study, positional cloning led to the identification of a gene encoding an RR1-type cuticular protein, BmorCPR2, highly expressed in larval chitin-rich tissues and at the mulberry leaf-eating stages, which is responsible for the silkworm stony mutant. In the Dazao-stony strain, the BmorCPR2 allele is a deletion mutation with significantly lower expression, compared to the wild-type Dazao strain. Dysfunctional BmorCPR2 in the stony mutant lost chitin binding ability, leading to reduced chitin content in larval cuticle, limitation of cuticle extension, abatement of cuticle tensile properties, and aberrant ratio between internodes and intersegmental folds. These variations induce a significant decrease in cuticle capacity to hold the growing internal organs in the larval development process, resulting in whole-body stiffness, tightness, and hardness, bulging intersegmental folds, and serious defects in larval adaptability. To our knowledge, this is the first study to report the corresponding phenotype of stony in insects caused by mutation of RR1-type cuticular protein. Our findings collectively shed light on the specific role of cuticular proteins in maintaining normal larval body shape and will aid in the development of pest control strategies for the management of Lepidoptera.

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.

[cDNA cloning, characterization and mRNA expression of a profilin from the swimming crab Portunus trituberculatus].

We isolated and characterized the profilin full-length cDNA from hemocytes of swimming crab Portunus trituberculatus. The profilin cDNA consists of 742 bp and the 375 bp open reading frame encodes a polypeptide of 125 amino acids, having a predicted isoelectric point of 5.87. The deduced amino acid sequence shows 42.9% amino acid sequence identity to the profilin of mosquito Anopheles gambiae. The profilin mRNA was highly expressed in hemocytes and moderately in hepatopancreas of normal crab. The higher expression of profilin mRNA is observed in crab challenged by the pathogenic bacterium Vibrio parahaemolyticus. These results suggest a potential role for profilin in pathogen host defense mechanisms.

Gene duplication and evidence for balancing selection acting on MHC class II DAA gene of the half-smooth tongue sole (Cynoglossus semilaevis).

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.