Antimicrobial peptides (AMPs) represent an efficient part of innate immunity and are found in a variety of life. Among them Histone 2A (H2A), as a promising class of AMPs, attracts great attention, but the in vivo mechanism of H2A derived AMP is still less known. Based on the acquisition of Sphistin, a synthetic 38-amino acid H2A derived peptide from Scylla paramamosain, as reported in our previous study, was truncated into three short fragments (Sph12-38, Sph20-38 and Sph30-38) and further investigated for its possible functional domains. The antimicrobial activities of these analogs against different Gram-positive bacteria, Gram-negative bacteria and fungi were illustrated. Among the analogs, Sph12-38 showed a stronger activity with a much lower minimum inhibitory concentration (3 μM) against Staphylococcus aureus, Corynebacterium glutamicum, Micrococcus lysodeikticus Fleming, Bacillus subtilis, Pseudomonas fluorescens, Aeromonas hydrophila and A. sobria in comparison with the reported Sphistin. A leakage of intracellular content was described in E. coli treated with Sph12-38. Unlike Sphistin which mainly disrupts the membrane integrity, Sph12-38 could also combine the A. sobria genomic DNA with a minimum concentration of 6 μM and was located intracellularly in cells observed under confocal laser scanning microscope imaging. In comparison with the control group of Oryzias melastigma injected with A. sobria alone, the group treated with a mixture of Sph12-38 and A. sobria showed a higher survival rate 7 days post-injection. Furthermore, in a pretreatment assay at 6 h, a higher survival rate was observed in the group injected with the mixture of Sph12-38 and A. sobria. Taken together, the synthetic peptide of Sph12-38 had a potent antimicrobial activity against bacteria. However, Sph12-38 had no cytotoxicity towards the hemolymph of S. paramamosain. Our study suggested that, as with Sph12-38, the H2A derived peptides were more likely prone to exert their activities in vivo through the truncated fragments while defending against different species of pathogens.

Comparative genomics research in non-model species has highlighted how invertebrate hosts possess complex diversified repertoires of immune molecules. The levels of diversification in particular immune gene families appear to differ between invertebrate lineages and even between species within lineages, reflecting differences not only in evolutionary histories, but also in life histories, environmental niches, and pathogen exposures. The goal of this research was to identify immune-related gene families experiencing high levels of diversification in eastern oysters, Crassostrea virginica. Families containing 1) transcripts differentially expressed in eastern oysters in response to bacterial challenge and 2) a larger number of transcripts compared to other species included those coding for the C1q and C-type lectin domain containing proteins (C1qDC and CTLDC), GTPase of the immune-associated proteins (GIMAP), scavenger receptors (SR), fibrinogen-C domain containing proteins (also known as FREPs), dopamine beta-hydrolase (DBH), interferon-inducible 44 (IFI44), serine protease inhibitors, apextrin, and dermatopontin. Phylogenetic analysis of two of the families significantly expanded in bivalves, IFI44 and GIMAP, showed a patchy distribution within both protostomes and deuterostomes, suggesting multiple independent losses and lineage-specific expansions. Increased availability of genomic information for a broader range of non-model species broadly distributed through vertebrate and invertebrate phyla will likely lead to improved knowledge on mechanisms of immune-gene diversification.

Histone H2A is known to participate in host immune defense through generating special antimicrobial peptides (AMPs), for which it has been an interesting research focus to characterize this kind of peptides in vertebrates and invertebrates. Although thousands of AMPs have been reported in variety of life species, only several AMPs are known in crabs and in particular no H2A-derived AMP has yet been reported. In the present study, a 38-amino acid peptide with antimicrobial activity was determined based on the sequence analysis of a histone H2A identified from the mud crab Scylla paramamosain. The histone H2A derived peptide was an AMP-like molecule and designated as Sphistin. Sphistin showed typical features of AMPs such as amphiphilic α-helical second structrue and positive charge net. The synthetic Sphistin exerted high antimicrobial activity against Gram-positive, Gram-negative bacteria and yeast, among which Aeromonas hydrophila, Pseudomonas fluorescens and Pseudomonas stutzeri are important aquatic pathogens. Leakage of the cell content and disruption of the cell surface were observed in bacterial cells treated with Sphistin using scanning electron microscopy. It was proved that the increasing cytoplasmic membrane permeability of Escherichia coli was caused by Sphistin. Further observation under confocal microscopy showed that Sphistin could combine onto the membrane of Staphylococcus aureus, E. coli MC1061 and Pichia pastoris but not translocate into the cytoplasm. Moreover, the affinity of Sphistin with either LPS or LTA was also testified that there was an interaction between Sphistin and cell membrane. Thus, the antimicrobial mechanism of this peptide likely exerted via adsorption and subsequently permeabilization of the bacterial cell membranes other than penetrating cell membrane. In addition, synthetic Sphistin exhibited no cytotoxicity to primary cultured crab haemolymphs and mammalian cells even at a high concentration of 100 μg/mL for 24 h. This is the first report of a histone-derived Sphistin identified from S. paramamosain with a specific antimicrobial activity and mechanism, which could be a new candidate for future application in aquaculture and veterinary medicine.

The effects of polycyclic aromatic hydrocarbons (PAHs) have been reported to modulate the immune response in aquatic animals, but the collected information of their effects on fish immunity is so far ambiguous. This study demonstrated that Benzo[ a ]pyrene (BaP) exposure altered the expression pattern of an antimicrobial peptide hepcidin (PM‐hepc) gene and the activities of some immune‐associated parameters in the lipopolysaccharide (LPS)‐challenged red sea bream ( Pagrus major ). It was observed that LPS could increase respiratory burst, lysozyme and antibacterial activity in P. major . However when the P. major was exposed to different concentrations of BaP (1, 4, or 8 μg L −1 ) for 14 days and then challenged with LPS there was no significant change in the lysozyme and antibacterial activity. It was further observed that LPS could induce the PM‐hepc mRNA expression at 3, 6, and 12‐h post‐LPS challenge. However, when P. major was exposed first to BaP for 14 days and then challenged with LPS, the expression of PM‐hepc mRNA was delayed in the liver until 24 h and not significantly induced until 48 and 96 h. The mRNA expression pattern was completely different from that only with LPS challenge, showing that BaP exposure changed the PM‐hepc mRNA expression pattern of fish with LPS challenge. This study demonstrated that BaP exposure can weaken or inhibit the induction of lysozyme and antibacterial activity in the LPS‐challenged P. major ; conversely BaP exposure could enhance the mRNA expression of PM‐hepc gene, indicating that the effect of BaP has different modulatory mechanism on hepcidin genes and immune‐associated parameters. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 517–525, 2014.

以大黄鱼(Pseudosciaena crocea)管家基因18S rRNA和β-actin作为内参基因,分别比较2个内参基因建立的相对定量曲线,最终确立以18S rRNA为参比基因,定量分析大黄鱼的Hepcidin抗菌肽基因.该相对定量分析方法所得结果与Northern-blot方法一致.应用建立的Hepcidin基因的实时荧光相对定量研究方法,对大黄鱼头肾中的Hepcidin基因转录物进行相对定量,为今后开展鱼体内免疫相关基因的表达特性、诱导机制等工作奠定研究基础.同时,克隆得到的大黄鱼β-actin基因和18S rRNA基因片段已提交基因库,并获得登录号.

Recently, two hepcidin variant genes (Om-hep1 and Om-hep2) were identified in a model fish marine medaka and both were highly induced in vivo with bacterial challenge, suggesting that the medaka hepcidin may have a similar function to other reported teleostean hepcidins. In the present study, the antibacterial, antiviral and antitumor activities of Om-hep1 were determined using its synthetic and recombinant pro-peptides. The recombinant pro-hepcidin1 was expressed in Escherichia coli and an effective method to produce recombinant Pro-Omhep1 was developed in order to obtain a right folded structure. The results showed that both the synthetic mature peptide and recombinant pro-peptide had similar antibacterial activity against Gram-positive and negative bacteria. In particular, both the synthetic mature Om-hep1 and recombinant Pro-Omhep1 inhibited the viral replication of white spot syndrome virus in the hematopoietic tissue cells of the crayfish Cherax quadricarinatus. Om-hep1 also presented antitumor activity on the cultured human hepatocellular carcinoma cells. In addition, the antimicrobial mechanism of Om-hep1 was measured and it was found that Om-hep1 was likely to be non-membranolytic. The recombinant Pro-Omhep1 performed better biological activity compared to the synthetic mature Om-hep1. This study suggested that Om-hep1 was likely to be an important multifunction protein involved in various resistance actions in the marine medaka immune system.

The gene expression patterns of CYP1A1 and Aryl hydrocarbon receptor(AhR2) of red seabream(Pagrus major) were both measured using real-time quantitative PCR(qPCR) when fish exposed to environmentally relevant concentration of BaP(0.1,0.5 and 1.0 μg/L,respectively).The results showed that CYP1A1 mRNA and AhR2 mRNA could be induced significantly,besides,the time of AhR2 mRNA induced ahead of the time of CYP1A mRNA induced.The two genes were induced markedly at the begining of BaP exposure,and then decreased to the basal levels after 72 h.The results demonstrate that BaP can regulate CYP1A1 and AhR2 transcript in a dose and time dependent manner.

The gene expression patterns of CYP1A1 and Aryl hydrocarbon receptor(AhR2) of red seabream(Pagrus major) were both measured using real-time quantitative PCR(qPCR) when fish exposed to environmentally relevant concentration of BaP(0.1,0.5 and 1.0 μg/L,respectively).The results showed that CYP1A1 mRNA and AhR2 mRNA could be induced significantly,besides,the time of AhR2 mRNA induced ahead of the time of CYP1A mRNA induced.The two genes were induced markedly at the begining of BaP exposure,and then decreased to the basal levels after 72 h.The results demonstrate that BaP can regulate CYP1A1 and AhR2 transcript in a dose and time dependent manner.