Ying Qiao

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

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

Molecular characterization and expression analysis of interferon-gamma in the large yellow croaker Larimichthys crocea.

The large yellow croaker Larimichthys crocea is an important mariculture fish species in China, and the bacterium Vibrio harveyi (V. harveyi) and the ciliate protozoan Cryptocaryon irritans (C. irritans) are the two major pathogens in its aquaculture sector. Interferon-gamma (IFN-γ) plays important roles in regulating both innate and cell mediated immune responses as an inflammatory cytokine. In this study, we obtained the nucleotide sequence of IFN-γ from the large yellow croaker (LcIFN-γ). The phylogenetic relationship tree of 18 available IFN-γ genes was constructed based on their sequences. Expression analyses in 10 various tissues were conducted after the croaker challenged with V. harveyi and C. irritans, respectively. Real time PCR analysis showed that the expression of LcIFN-γ was observed broadly in health individuals. After injected with V. harveyi, the 10 tissues had a higher expression of IFN-γ at the first day (1 d); only spleen, muscle, intestine, heart and skin had higher expressions after infected with C. irritans at 1 d. Major immune tissues (skin, gill, head kidney and spleen) and detoxification tissue (liver) were sampled at 0 h, 6 h, 1 d, 2 d, 3 d, 4 d, 5 d and 7 d to understand the expression trends of LcIFN-γ after challenged with C. irritans. The expressions of LcIFN-γ in skin and gill (the primary immune organs) showed a clear correlative relationship with the life cycle of C. irritans.

Discovery and molecular cloning of piscidin-5-like gene from the large yellow croaker (Larimichthys crocea)

The large yellow croaker (Larimichthys crocea) (Perciformes: Sciaenidae), is a commercially important mariculture fish in southeastern China. Because of the poor managements and water deterioration in the mariculture areas, various diseases have been out-broken frequently. The disease such as the Cryptocaryonosis, caused by a ciliate protozoan Cryptocaryon irritans [1], can lead to extreme high mortality and significant economic loss for the large yellow croaker and some other cultured fishes [2e4]. To prevent and control fish diseases, particularly to those parasitic diseases, chemical medicines have been commonly used [5,6]. However, such treatments may have long-term influences on aquatic environment and human health simultaneously [7e9]. Therefore, more environmental friendly treatments and approaches for fish disease are urgently needed. Fishes mainly rely on their innate immune system to resist exogenous and endogenous pathogens [10,11], and antimicrobial peptides (AMPs) are known to be potent and effective components, which play an important role in resists pathogens infection [12e18]. A novel and notable antimicrobial peptides family of fish is piscidin [9,19], which was initially isolated from the mast cells of hybrid striped bass (Morone saxatilis Morone chrysops) in 2001 [20]. Most piscidin members are usually less than 26 amino acids

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

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

Identification and molecular characterization of Cathepsin L gene and its expression analysis during early ontogenetic development of kuruma shrimp Marsupenaeus japonicus

Cathepsin L gene is a member of the cysteine proteinase gene group. In this study Cathepsin L gene was isolated from Kuruma shrimp Marsupenaeus japonicus (Mj-Cathepsin L) and the full-length DNA sequence was 1 963 bp. Mj-Cathepsin L protein showed high homologies with other Cathepsin L proteins documented in vertebrates, mollusks and other crustaceans. Expression analysis of Mj-Cathepsin L gene in different tissues revealed that it was predominant in hepatopancreas. During early ontogenetic development stages Mj-Cathepsin L showed a development-regulated expression, and the Mj-Cathepsin L showed a molting stage-regulated expression during the five molting stages, inferring its role in the ontogenic development of M. japonicus. Two kinds of forms of Mj-Cathepsin L protein: pro-Cathepsin L and Cathepsin L were measured in hepatopancreas, stomach and intestine by Western Blotting.

Pathogenic bacterium Vibrio harveyi: an endosymbiont in the marine parasitic ciliate protozoan Cryptocaryon irritans

Vibrio harveyi, known as a pathogenic bacterium caused severe secondary bacterial infections of the large yellow croaker Larimichthys crocea, was identified as an endosymbiont in the marine parasitic ciliate protozoan Cryptocaryon irritans. Meta 16S sequencing method was used to identify the bacterial flora in C. irritans, and V. harveyi was isolated via culture-dependent method. Vibrio harveyi was observed in cytoplasm of C. irritans at the stage of tomont both by transmission electron microscopy and by Fluorescence in situ hybridization; no signal, however, was detected in nucleus area. The relationship between V. harveyi and C. irritans and the role of endosymbiotic V. harveyi in C. irritans merit further investigation.